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Full text of "AOAC: Official Methods of Analysis (Volume 1)"

*********A********* 





By Authority Of 

THE UNITED STATES OF AMERICA 

Legally Binding Document 



By the Authority Vested By Part 5 of the United States Code § 552(a) and 
Part 1 of the Code of Regulations § 51 the attached document has been duly 
INCORPORATED BY REFERENCE and shall be considered legally 
binding upon all citizens and residents of the United States of America. 
HEED THIS NOTICE : Criminal penalties may apply for noncompliance. 




Document Name: AOAC: Official Methods of Analysis (Volume 1) 



CFR Section(s): 9 CFR 318.19(b) 



Standards Body: AOAC International 



OFFICIAL 
ETHODS of 
ANALYSIS 

15 th Edition, 1990 



ASSOCIATION 
of OFFICIAL 
ANALYTICAL 
CHEMISTS 



Agricultural Chemicals; 
Contaminants; Drugs 

VOLUME ONE 



OFFICIAL 
METHODS OF ANALYSIS 

OF THE 

ASSOCIATION OF OFFICIAL 
ANALYTICAL CHEMISTS 

Edited by Kenneth Helrich 



FIFTEENTH EDITION, 1990 



Published by the 

Association of Official Analytical Chemists, Inc. 

Suite 400 

2200 Wilson Boulevard 

Arlington, Virginia 22201 USA 



Copyright © 1920, 1925, 1931, 1936, 1940, 1945, 1950, 1955, 1960, 1965 

by the Association of Official Agricultural Chemists 

and 1970, 1975, 1980, 1984, 1990 

by the Association of Official Analytical Chemists 

The methods of the Association were also copyrighted in 1916, when they were 
published in the Journal of the Association of Official Agricultural Chemists, 

All rights reserved. No part of this book may be reproduced in any form or by 
any means without the written permission of the Association. 

Printed in the United States of America 

Printed on acid-free paper 

Library of Congress Catalog Card Number 85-647589 
ISBN 0-935584-42-0 
ISSN 0066-96 IX 

A copy of the 15th edition of this publication is on file with the Office of the 
Federal Register. U.S. Government Agencies may apply to the Director of the 
Office of the Federal Register for approval to incorporate this edition by reference 
in their regulations. The procedures that Federal agencies must follow in applying 
for the Director's approval are in Title 1, Part 51, of the Code of Federal 
Regulations . 

iv 



Important Notices 

to Librarians and ASI Users 

of this Edition 



FREE SUPPLEMENTS 



Purchasers of this volume will receive the annual supplements of Changes in Official Methods of Analysis without 
additional charge only by removing the card inserted after p. 40, filling in the specific name and address to which 
the supplements should be sent, and returning this card to the Association. Address must include postal code or delivery 
cannot be assured. 

SURPLUS METHODS 

Neither the 10th, 11th, 12th, 13th, or 14th editions should be destroyed upon appearance of the 15th edition. They 
contain surplus methods which are not reprinted in the 15th edition. See page xvii for the definition of surplus methods. 
If methods marked with this symbol, *, continue to be used and therefore should be retained in full, please notify 
the Association. 

USE OF METHODS 

Analytical methods and procedures included in this volume are those which AOAC members have evaluated and 
validated through collaborative studies and appropriate similar techniques to give accurate and reproducible analytical 
results on the matrix and analyte to which the method is stated to apply, provided the analysis is conducted by a 
competent analyst as written. No warranty, implied or expressed, is made by the Association on methods described 
or products mentioned. The mention of commercial trade names does not imply endorsement by AOAC or its members 
over similar products that might be suitable. AOAC and its members who have aided in development and validation 
of methods enclosed assume no responsibility for any economic, personal injury, or other damage that may occur to 
individuals or organizations because of use of such methods. 

EQUIVALENT COMMERCIAL PRODUCTS 

AOAC recognizes that the instrumentation and laboratory supplies stated in these published methods may have 
equivalents which will produce analytical results having the same statistical performance. Names of manufacturers 
and suppliers and trade names are furnished solely as a matter of identification and convenience within the context 
of the way each method was developed within the originator's laboratories, without implication of AOAC promotion, 
approval, endorsement, or certification. The same or equivalent products, instruments, supplies, apparatus, or reagents 
available from suppliers other than those named or other brands from other sources may serve equally well if proper 
validation indicates that their use is satisfactory. 

INQUIRIES 

Inquiries regarding procedures published in this book should be directed to AOAC, Executive Director, 2200 Wilson 
Blvd, Arlington, VA 22201-3301 USA. Telephone (703) 522-3032. Facsimile (703) 522-5468. 

Inquiries regarding purchase of Official Methods of Analysis, supplements ("Changes in Official Methods"), Journal 
of the AOAC, or other AOAC publications should be directed to AOAC, Fulfillment Coordinator, 2200 Wilson Blvd, 
Arlington; VA 22201-3301 USA. Telephone (703) 522-3032. Facsimile (703) 522-5468. 



COMMENTS ON METHODS 

AOAC adopts methods that show by their performance data, obtained through the collaborative study, what can be 
expected of them. As analysts use AOAC methods, they generate additional information and data concerning the 
applicability, specificity, sensitivity, and accuracy of the methods. Analysts are requested to advise AOAC about their 
experiences with official methods published in this book. In particular, analysts should notify AOAC of problems in 
the performance of any method that indicate the method should be revised or restudied. Direct comments to AOAC, 
Assistant Executive Director, Technical Services and Development, 2200 Wilson Blvd, Arlington, VA 22201-3301 
USA. Telephone (703) 522-3032. Facsimile (703) 522-5468. 



Preface to the 15th Edition 

The most obvious change in this new edition of Official Methods of Analysis of the AOAC is the new format, 
splitting the original single volume into two volumes containing Agricultural Chemicals, Contaminants, and Drugs in 
Volume I, and Food Composition in Volume II. Extensive discussions, user polls, and committee deliberations re- 
garding the most useful and desirable form for publication of the 15th Edition led to the decision to make this change. 
While this has necessitated the repetition of a few items such as the index and the safety chapter, the convenience of 
smaller volumes with a logical division of subject matter is a definite advantage. The two volume arrangement also 
allows for more manageable growth as the number of validated methods increases. 

In actual content, the most striking change in this new edition is the assignment of permanent numbers to all official 
methods. This tedious and time consuming task was undertaken primarily because, as Editor William Horwitz stated 
in the preface to the Thirteenth Edition in 1980, "Users expressed a desire for a system that will keep the same 
reference number of a given method from edition to edition." There are significant advantages to a permanent num- 
bering system. Since AOAC methods are cited worldwide in laws and regulations at every level of government, in 
definitions of standards of identity, and in public and private specifications and contracts, it is practical and highly 
desirable to have a single, unchanging number for any method. Permanent numbers will reduce citation errors and 
simplify citation by eliminating the necessity to specify editions in some instances. The publication of future editions 
will not be encumbered by the need to keep track of changing numbers for existing, unchanged methods. There is 
also the advantage of desirable consistency for electronic databases. 

Permanent numbers are based on the year the method first appeared in "Changes in Official Methods of Analysis" 
in the Journal. The year determines the first three numbers with the next digits being simply the sequence in which 
the methods were adopted in a given year. For example, the first method adopted in 1988 and published in "Changes 
in Official Methods of Analysis" in 1989 would be given the number 989.01. The year of adoption was not researched 
for methods adopted before 1960. For those, the numbers are based on the date of the first reference, or, if that was 
not available, on the year the method first appeared in Official Methods of Analysis of the AOAC. An index to the 
new numbers is included to facilitate locating methods when only the method number is known. References for the 
more recent methods in the 15th Edition have been verified, corrected, and brought up to date so that the user can 
more readily find the original work that resulted in adoption of the method. The list of suppliers, as well as supplier 
references in each method, has also been revised and updated. 

Method performance data appear at the beginning of methods adopted as part of "Changes in Official Methods of 
Analysis" in 1989. Previously published method performance data (14th Edition) have been deleted because of a 
change in the procedure for calculation. All future new methods will have the method performance section included, 
using the performance parameters that were adopted by the AOAC Board of Directors in 1988. Method performance 
data are generated from the collaborative study results. 

The addition of about 150 new methods to the 15th Edition continues to respond to the AOAC mandate to keep 
pace with the practical needs of regulatory and research chemists and microbiologists. Some previously adopted meth- 
ods have been expanded in scope; some have had efficiency or accuracy improved. Additional methods have been 
declared surplus and omitted from the present Edition in instances where they were no longer sufficiently used to 
warrant reprinting. Again, the user is asked to preserve previous editions for the rare instance when a surplus method 
may be needed. 

Liquid chromatography (LC) and gas chromatography (GC) have continued to be the most popular and useful 
techniques of analysis. A variety of detectors are still being utilized, along with internal standards. In addition to 
sophisticated modern instrumentation, such classical techniques as gravimetric analysis, distillation and physical sep- 
arations, and Kjeldahl nitrogen determinations are still yielding new, needed methods. 

Among the most innovative techniques are the additions to the chapter on Microbiological Methods. Over twenty 
new methods have been added to this chapter. These include DNA colony hybridization, enzyme immunoassay, mi- 
crobial receptor assay, and immunodiffusion methods. Many new methods utilize beads, pretreated pectin gel films, 
dry rehydratable films, and hydrophobic grid plates. Sources of the kits and pertinent information on components are 
provided. In some instances, generic substitutions are possible for kit components. 

As always, thanks are due to the many individuals who worked so diligently and carefully to maintain the quality 



of the Official Methods of Analysis of the AOAC. These include the Associate Referees, General Referees, collabo- 
rators, and Methods Committee members who researched, perfected, validated, and reviewed each method. The Gen- 
eral Referees, beyond the call of duty, contributed their services as Chapter Associate Editors, arranging and reviewing 
chapters where their expertise was an invaluable asset. The AOAC Official Methods Board, Editorial Board, and 
Board of Directors provided the guidance throughout the duration of this project. The AOAC Scientific Publications 
staff did a heroic job from the very beginning in editing methods, keeping the methods publication on schedule, 
handling the unending details of actual publication, and renumbering over 1,000 pages of methods with innumerable 
cross references. 

Kenneth Helrich 

Editor, Official Methods of Analysis 



Contents 



Volume S 



PAGE 

Important Notices v 

Preface vii 

About the Association xiii 

Guide to Method Format xiv 

Definitions of Terms and Explanatory 

Notes xv 

Collaborative Study Procedures xxii 

CHAPTER 

1. Agricultural Liming Materials 1 

Calcium Silicate Slags 3 

Gravimetric Elemental Analyses 4 

Chelometric Elemental Analyses 5 

Colorimetric Elemental Analyses 6 

2. Fertilizers 9 

Water 11 

Phosphorus 12 

Nitrogen 17 

Potassium 23 

Other Elements 27 

Peat 36 

3. Plants 40 

Metals 40 

Individual Metals 43 

Nonmetals 50 

Other Constituents 58 

Pigments 62 

Tobacco 64 

4. Animal Feed 69 

Protein 70 

Urea 76 

Nitrogen 77 

Fat 79 

Fiber 80 

Sugars 83 

Minerals 84 

Microscopy 88 

5. Drugs in Feeds 91 

Chemical Methods 91 

Microbiological Methods 

for Antibiotics 115 

Chemical Methods for Antibiotics 129 



PAGE 

Common and Chemical Names 

of Drugs 132 

6. Disinfectants 133 

Phenol Coefficient Methods 133 

Use-Dilution Methods 135 

Other Tests 137 

7. Pesticide Formulations 147 

General Methods 147 

Inorganic and Organometallic 

Pesticides and Adjuvants 153 

Pesticides Related to Natural 

Products and Their Synergists 164 

Organohalogen Pesticides 174 

Thiophosphorus and Other 

Organophosphorus Pesticides 197 

Carbamate, Substituted Urea, 

and Triazine Pesticides 212 

Miscellaneous Pesticides 223 

Common and Chemical Names 

of Pesticides 230 

8. Hazardous Substances 232 

9. Metals and Other Elements 

at Trace Levels in Foods 237 

Multielement Methods 237 

Single Element Methods 242 

10. Pesticide and Industrial 

Chemical Residues 274 

Multiresidue General 

Considerations 274 

Organochlorine Residues 283 

Organophosphorus Residues 286 

Fumigant Residues 290 

Carbamate Residues 291 

Individual Residues 294 

Common and Chemical Names 

of Pesticides 310 

11. Waters; and Salt 312 

Waters 312 

Salt 334 

12. Microchemical Methods 337 



PAGE 

13. Radioactivity 349 

14. Veterinary Analytical Toxicology 356 

15. Cosmetics 359 

General Methods 359 

Deodorants and Antiperspirants 361 

Dipilatories 365 

Face Powders 365 

Hair Preparations 366 

Suntan Preparations 367 

16. Extraneous Materials: Isolation 369 

General 369 

Beverages and Beverage 

Materials 373 

Dairy Products 375 

Nuts and Nut Products 379 

Grains and Their Products 380 

Baked Goods 385 

Breakfast Cereals 387 

Eggs and Egg Products 388 

Poultry, Meat, and Fish and Other 

Marine Products 389 

Fruits and Fruit Products 391 

Snack Food Products 393 

Sugars and Sugar Products 393 

Vegetables and Vegetable 

Products 394 

Spices and Other Condiments 397 

Miscellaneous 406 

Animal Excretions 407 

Mold and Rot Fragments 416 

Fruits and Fruit Products 418 

Vegetables and Vegetables 

Products 419 

General References 423 

17. Microbiological Methods 425 

Cross Reference Tables 425 

Eggs and Egg Products 427 

Chilled, Frozen, Precooked, or 

Prepared Foods, and Nutmeats 429 

Coliforms 434 

Escherichia coll 438 

Staphylococcus 449 

Sterility (Commercial) of Foods 

(Canned, Low Acid) 455 

Clostridium 459 

Bacillus 464 

Salmonella 467 

Vibrio 492 

Miscellaneous 494 

Somatic Cells 496 

18. Drugs: Part 1 498 

Solvents 498 

Halogenated Drugs 499 



PAGE 

Inorganic Drugs 501 

Antihistamines 512 

Alkanolamines 515 

Phenethylamines 520 

Aminobenzoates 521 

Synthetics 525 

Microchemical Tests 533 

Microscopy 541 

Miscellaneous 542 

Common and Chemical 

Names of Drugs 545 

19. Drugs: Part II 548 

Acids 548 

Phenolic Drugs 549 

Analgesics and Antipyretics 553 

Hypnotics and Sedatives 558 

Anticoagulants 565 

Sulfonamides 567 

Thiazides 570 

Other Sulfur-Containing Drugs 573 

Common and Chemical 

Names of Drugs 578 

20. Drugs: Part III 579 

Opium Alkaloids 579 

Tropane Alkaloids 583 

Xanthine Alkaloids 584 

Ipecac Alkaloids 584 

Ephedra Alkalids 586 

Ergot Alkaloids 588 

Physostigmine Alkaloids 590 

Chinchona Alkaloids 592 

Rauwolfia Alkaloids 593 

Other Alkaloids 598 

Digitalis 599 

Other Natural Products 602 

Common and Chemical 

Names of Drugs 606 

21. Drugs: Part IV 607 

Natural Estrogens 607 

Synthetic Estrogens 609 

Progestational Steroids 612 

Adrenocortico Steroids 613 

Thyroid 618 

Common and Chemical 

Names of Drugs 619 

22. Drugs: Part V 620 

Common and Chemical Names of Drugs 624 



23. Drugs and Feed Additives in Animal 

Tissues 625 

Common and Chemical Names of 

Drugs 636 



PAGE 

24. Forensic Sciences 637 

Appendix: Standard Solutions and Certified 

Reference Materials 640 

Appendix: Laboratory Safety 649 

Appendix: Reference Tables 656 



PAGE 

Appendix: Guidelines for Collaborative Study 
Procedure to Validate 
Characteristics of a Method 
of Analysis 673 

Subject Index 1-1 

Method Number Index 1-55 



Volume II 



PAGE 

Important Notices v 

Definitions of Terms and Explanatory 

Notes xi 

Guide to Method Format xviii 

CHAPTER 

25. Baking Powders and Baking Chemicals . . 685 

26. Distilled Liquors 690 

Spirits 690 

Cordials and Liqueurs . . . . 704 

27. Malt Beverages and Brewing Materials . . 708 

Beer 708 

Barley 723 

Malt 723 

Cereal Adjuncts 730 

Hops . . .! 731 

Brewing Sugars and Sirups 733 

Wort 734 

Yeast 735 

Brewers' Grains 737 

28. Wines 739 

General 739 

Preservatives 749 

Flavors 750 

29. Nonalcoholic Beverages and Concentrates 751 

30. Coffee and Tea 757 

Green Coffee 757 

Roasted Coffee 757 

Tea 761 

31. Cacao Bean and Its Products 763 

General 763 

Shell 764 

Chocolate Liquor 770 

Fat 770 

Other Constituents 772 



PAGE 

32. Cereal Foods 777 

Wheat Flour 777 

Wheat, Rye, Oats, Corn, Buckwheat, 
Rice, Barley, and Soybeans and Their 

Products Except Cereal Adjuncts 788 

Bread 790 

Baked Products 794 

Macaroni, Egg Noodles, 

and Similar Products 796 

33. Dairy Products . . 802 

Sampling 802 

Milk 804 

Cream 831 

Evaporated and Condensed Milk 833 

Dried Milk, Nonfat Dry Milk, 

and Malted Milk 834 

Butter 836 

Cheese 840 

Ice Cream and Frozen Desserts 850 

34. Eggs and Egg Products 853 

35. Fish and Other Marine Products 864 

36. Flavors 890 

Vanilla Extract and Its Substitutes 890 

Lemon, Orange, and Lime Extracts, 

Flavors and Oils 898 

Almond Extract 903 

Cassia, Cinnamon, and Clove Extracts. . . 905 

Flavor Extracts and Toilet Preparations . . 905 

37. Fruits and Fruit Products 910 

38. Gelatin, Dessert Preparations, and Mixes 929 

39. Meat and Meat Products 931 

40. Nuts and Nut Products 949 



PAGE 

41. Oils and Fats 951 

42. Vegetable Products, Processed 987 

Canned Vegetables 987 

Dried Vegetables 994 

Frozen Vegetables 995 

43. Spices and Other Condiments 999 

44. Sugars and Sugar Products 1010 

Sugars and Sirups 1010 

Molasses and Molasses Products 1021 

Confectionary 1024 

Honey 1025 

Maple Sap, Maple Sirup, Maple 

Sirup Products 1034 

Sugar Beets 1039 

Corn Sirups and Sugars 1039 

45. Vitamins and Other Nutrients 1045 

Chemical Methods 1045 

Microbiological Methods 1080 

Bioassay Methods 1091 

Nutritionally Related Components 1099 

Infant Formula 1 106 

46. Color Additives 1115 

Separation and Identification of Color 

Additives in Foods, Drugs, 

and Cosmetics 1115 

Intermediates 1 126 

Subsidiary and Lower Sufonated Dyes ... 1132 

Metals and Other Elements 1132 



PAGE 

Halogens 1135 

Miscellaneous 1 1 36 

47. Food Additives: Direct 1 137 

Antioxidants 1 137 

Chemical Preservatives 1 141 

Emulsifying Agents . 1 163 

Enzymes 1 1 64 

Miscellaneous 1 165 

48. Food Additives: Indirect 1 176 

49. Natural Poisons 1 1 84 

Mycotoxins-General 1 1 84 

Aflatoxins 1185 

Aflatoxin M 1199 

Deoxynivalenol 1205 

Ochratoxins 1207 

Patulin 1209 

Sterigmatocystin 1210 

Zearalenone 1211 

Marine Toxins 1213 

Phytotoxins 1213 

Appendix: Standard Solutions and Certified 

Reference Materials 1214 

Appendix: Laboratory Safety 1223 

Appendix: Reference Tables 1230 

Subject Index 1-1 

Method Number Index 1-55 



About the Association 



PURPOSE AND FUNCTION 



AWARDS 



The primary objectives of the Association of Official Ana- 
lytical Chemists (AOAC) are to obtain, improve, develop, test, 
and adopt uniform, precise, and accurate methods for the anal- 
ysis of foods, drugs, feeds, fertilizers, pesticides, water, or 
any other substances affecting public health and safety, eco- 
nomic protection of the consumer, or quality of the environ- 
ment; to promote uniformity and reliability in the statement of 
analytical results; to promote, conduct, and encourage research 
in the analytical sciences related to agriculture and public health 
and the regulatory control of commodities in these fields; and 
to afford opportunity for discussion of matters of interest to 
scientists engaged in relevant pursuits. 

AOAC itself maintains no laboratories, conducts no anal- 
yses, performs no tests. The actual work of devising and test- 
ing methods is done by members of AOAC in their official 
and professional capacities as staff scientists of federal, state, 
provincial, and municipal regulatory agencies, experiment sta- 
tions, colleges and universities, commercial firms, and con- 
sulting laboratories. 

AOAC coordinates these scientific studies, receives and 
evaluates the results, gives official sanction to acceptable 
methods, and publishes and disseminates the methods. 

The reliability of methods of analysis is more important than 
ever before. Regulatory agencies need reliable, reproducible, 
and practical methods to enforce laws and regulations. Indus- 
try needs reliable methods to meet compliance and quality con- 
trol requirements. Few organizations in the world are devoted 
primarily to testing and validating analytical methods through 
interlaboratory collaborative studies — as is AOAC. 



MEETINGS 

The Annual International Meeting is the focal point of 
AOAC's yearly work. Here, members have opportunities to 
exchange ideas with colleagues from all over the world, and 
to update their technical knowledge at scientific sessions and 
symposia, exhibits, and short courses. 

The regional section program provides AOAC-affiliated lo- 
cal or regional scientific meetings, workshops, short courses, 
and other activities for laboratory analysts. Each regional sec- 
tion is organized by a local volunteer committee. 



COOPERATIVE ACTIVITIES 

AOAC has established joint committees, liaison, and rep- 
resentation with numerous scientific organizations worldwide . 
Thus, methods are often developed in cooperation with other 
standards- setting bodies. AOAC liaison representatives for 
contact outside North America are the following: Derek Ab- 
bott, 33 Agates Ln, Ashtead, Surrey KT21 2ND, England; 
Lars Appelqvist, Swedish University of Agricultural Sciences, 
Dept of Food Hygiene, S 750 07 Uppsala, Sweden; and Mar- 
greet Lauwaars, PO Box 153, 6720 AD Bennekom, The Neth- 
erlands. 



The awards program of AOAC includes the following: The 
Scholarship Award is given each year to a student intending 
to do further study or work in an area important to public health 
or agriculture. 

The Fellow of the AOAC Award is given to selected mem- 
bers in recognition of at least 10 years of meritorious service 
to the Association as referees and/or committee members. 

The Harvey W. Wiley Award, honoring the "father" of the 
U.S. Pure Food and Drug Act and a founder of AOAC, is 
presented each year to a scientist or group of scientists who 
have made outstanding contributions to analytical methodol- 
ogy in an area of interest to AOAC. The $2500 award is sup- 
ported by the Wiley Fund. 

PUBLICATIONS 

Official Methods of Analysis includes full details of official 
methods but no descriptive or interpretative material or tables 
of data. However, AOAC publishes the Journal of the AOAC, 
which contains research articles and reports of the develop- 
ment, validation, and interpretation of analytical methods, and 
all collaborative study results. Journal contributors and its 
readers represent the worldwide analytical science community. 
The Journal is a forum for the exchange of information among 
methods researchers. The Journal also records the transactions 
of the Annual International Meeting, including committee and 
referee reports, lists of officers, referees, and committee mem- 
bers, and all official actions of the Association, including newly 
adopted methods. The Association publishes a variety of other 
books, manuals, video tapes, and symposium proceedings of 
interest to analytical scientists. 

MEMBERSHIP 

The organization of AOAC consists of the members; the Board 
of Directors, a governing body concerned with administration 
and policy making; Official Methods Board; Editorial Board; 
special standing committees and other groups concerned with 
development of methods and general activities; and the head- 
quarters staff which carries out the publications program and 
manages the Association. 

The AOAC Bylaws provide for individual members and sus- 
taining members. Chemists, microbiologists, and other sci- 
entists engaged in analysis or analytical research related to ag- 
riculture and public health, and employed by a college or 
university, any agency of a local, state, provincial, or national 
government, or firm or industry concerned with commodities 
or substances of interest to AOAC may be members. Sustain- 
ing members are government agencies or private industries that 
provide financial support to AOAC. 

The Referee, published 12 times yearly, is sent free to all 
members, and contains news about methods, collaborative 
studies, meetings, publications, AOAC people, board and 
committee activities, regional sections, and other items of in- 
terest. All members also receive the membership directory, 
issued annually. 



XIII 



GUIDE TO METHOD FORMAT* 



Unique number identifies 

method by year of 

adoption or first 

appearance in Official 

Methods of Analysis 

(older methods). 

980= first action 1980; 

.06= sequence of 

adoption in 1980. 



Chemical names of 

pesticides and drugs are 

given at end of pertinent 

chapter. 



Cautionary notes refer 
to Safety Chapter. 



Addresses for suppliers 

frequently cited 

throughout book are 

listed in "Definitions of 

Terms and Explanatory 

Notes." 



Letters identify main 
sections for ease of 
citation and cross- 
referencing. 



Calculation symbols are 

identified and show 
correct units. 



Chemical Abstracts 

Service Registry 

Number. A unique 

identifier that may be 

used to search a number 

of data-retrieval systems, 



-980.06 Captan in Pesticide Formulations 

Liquid Chromatographic Method - 
First Action 1980 
Final Action 1982 

AOAC-CIPAC Method 



(Method is suitable for tech. captan and formulations with captan as- 
only active ingredient.) 

A. Principle 

— Captan is extd from inerts with soln of diethyl phthalate in CH 2 Cl 2 . 
Soln ischromatgd on microparticulate silica gel column, using CH 2 C1 2 _ 
as mobile phase. Ratio of captan peak ht to diethyl phthalate peak ht is 
calcd from UV response and compared to std material for quantitation . 



(Caution: See safety notes on pipets and pesticides.) 

8. Apparatus and Reagents 

(a) Liquid chromato graph. — Able to generate over 1000 psi and ■ 
measured at 254 nm, 



— (d) Diethyl phthalate.— EM Science No. 1295. 

(e) Reference std captan. — Chevron Chemical Co., PO Box 4010, 
Richmond, CA 94804. 



C. Preparation of Standard 

(a) Internal std soln. — 0.312 mg diethyl phthalate/mL. Weigh 
ca 156 mg diethyl phthalate and transfer to 500 mL vol. flask. Dil. to 
vol. with same CH 2 C1 2 to be used for mobile . . . within 20%. 

-D. Preparation of Sample 

Accurately weigh sample expected to contain 40 mg captan into 
glass bottle. Pipet in 50 mL internal std soln. Place on mech. shaker 
15 min. Centrf. and filter supernate thru glass fiber paper. Prep, fresh 
sample daily. 

£. Determination 

Adjust operating parameters to cause captan to elute in 4-6 min. 
Maintain all parameters const thruout analysis. Typical values are: 
flow rate, 2.5 rnLCH 2 Cl 2 /min, max.; pressure, ca 800 psi; chart speed - 
0.2'7min; mobile phase . . . immediately preceding and following 
sample injections must agree to within ± 2% of their mean. If not, 
repeat detn. 

F. Calculation 

Measure peak hts to 3 significant figures, and calc. ratio for each 
injection. Average 4 std ratios, and the 2 sample ratios. 

% Captan = (R/R') x (W'(W) x P 

where R ~ av. sample ratio (captan peak ht/diethyl phthalate peak ht); 
R' = av. std ratio (captan peak ht/diethyl phthalate peak ht); W — mg 
sample; W ~ mg std, andP - % purity of std. 

Ref.: JAOAC 63, 1231(1980). 

-CAS-133-06-2 (captan) 



Method head may 

include analyte and 
matrix, type of method, 
official status, 
cooperating organization. 

Applicability 
statement— limitations 

on use of method or other 
information. 



Scientific basis for 

method of analysis. 



Specifications for 

necessary laboratory 
apparatus and reagent 
preparations. See also 
"Definitions of Terms and 
Explanatory Notes." 



Method may be divided 
into several descriptive 
sections. 



Abbreviations used 

throughout method are 
defined in "Definitions of 
Terms and Explanatory 
Notes." 



References direct the 
user to the published 
collaborative study and 
any subsequent revisions 
in the method. Other 
informative references 
may be included. 



* Method shown is incomplete to allow space for description. 



Definition of Terms and Explanatory Notes 



Official Methods 

(1) Official methods are designated first action or final ac- 
tion, and, in a few cases, procedures. A first action method 
has undergone collaborative study, has been recommended by 
the appropriate General Referee and Methods Committee, has 
been approved interim first action by the chairman of the Of- 
ficial Methods Board, and has been adopted official by the 
Association members at an annual meeting. A method may be 
adopted final action a minimum of 2 years after it has been 
adopted first action, and, again, after it has been recommended 
by the appropriate General Referee and Methods Committee 
and voted on by the Association members at an annual meet- 
ing. 

A sampling or sample preparation procedure or other type 
of procedure for which an inter! aboratory collaborative study 
is impractical may be adopted, as above, as a procedure. 

All methods in this book — first action, final action, or pro- 
cedure — are official methods of AOAC. 

Reagents 

(2) Term "H 2 CT means distilled water, except where oth- 
erwise specified, and except where the water does not mix 
with the determination, as in "H 2 bath." 

(3) Term "alcohol" means 95% ethanol by volume. Alco- 
hol of strength x% may be prepared by diluting x mL 95% 
alcohol to 95 mL with H 2 0. Absolute alcohol is 99.5% by 
volume. Formulas of specially denatured alcohols (SDA) used 
as reagents are as follows: 



SDA No. 


100 


parts alcohol plus 


1 


5 


wood alcohol 


2-B 


0.5 


benzene or rubber 
hydrocarbon solv. 


3-A 


5 


MeOH 


12-A 


5 


benzene 


13-A 


10 


ether 


23-A 


10 


acetone 


30 


10 


MeOH 



"Reagent" alcohol is 95 parts SDA 3-A plus 5 parts isopro- 
panol. 

(4) Term "ether" means ethyl ether, peroxide-free by fol- 
lowing test: To 420 mL ether in separator add 9.0 mL 1% 
NH4VO3 in H 2 S0 4 (1 + 16). Shake 3 min and let separate. 
Drain lower layer into 25 mL glass-stoppered graduate, dilute 
to 10 mL with H 2 S0 4 (1 + 16), and mix. Any orange color 
should not exceed that produced by 0.30 mg H 2 2 (1 mL of 
solution prepared by diluting 1 mL 30% H 2 2 to 100 mL with 
H 2 0) and 9.0 mL 1% NH4VO3 in H 2 S0 4 (1 + 16). Peroxides 
may be eliminated by passing <700 mL ether through 10 cm 
column of Woelm basic alumina in 22 mm id tube. 

(5) Reagents listed below, unless otherwise specified, have 
approximate strength stated and conform in purity with Rec- 
ommended Specifications for Analytical Reagent Chemicals of 
the American Chemical Society: 



Assay 

Sulfuric acid 95.0-98.0% H 2 S0 4 

Hydrochloric acid 36.5-38.0% HCI 

Nitric acid 69.0-71 .0% HNQ 3 

Fuming nitric acid >90% HN0 3 

Acetic acid >99.7% HC 2 H 3 2 

Hydrobromic acid 47.0-49.0% HBr 

Ammonium hydroxide 28-30% NH 3 

Phosphoric acid >85% H 3 P0 4 



Where no indication of dilution is given, reagent concentration 
is the concentration given above. 

(6) All other reagents and test solutions, unless otherwise 
described in the text, conform to requirements of the American 
Chemical Society. Where such specifications have not been 
prepared, use highest grade reagent. When anhydrous salt is 
intended, it is so stated; otherwise the crystallized product is 
meant. 

(7) Unless otherwise specified, phenolphthalein (phthln) used 
as indicator is 1% alcohol solution; methyl orange is 0.1% 
aqueous solution; methyl red is 0.1% alcohol solution. 

(8) Directions for standardizing reagents are given in the 
chapter on Standard Solutions and Certified Reference Mate- 
rials. 

(9) Unusual reagents not mentioned in reagent sections or 
cross referenced, other than common reagents normally found 
in laboratory, are italicized the first time they occur in a method. 

(10) Commercially prepared reagent solutions must be 
checked for applicability to specific method. They may con- 
tain undeclared buffers, preservatives, chelating agents, etc. 

(11) In expressions (1 + 2), (5 + 4), etc., used in connec- 
tion with name of reagent, first numeral indicates volume re- 
agent used, and second numeral indicates volume of H 2 0. For 
example, HCI (1 + 2) means reagent prepared by mixing 1 
volume of HCI with 2 volumes of H 2 0. When one of the re- 
agents is a solid, expression means parts by weight, first nu- 
meral representing solid reagent and second numeral H 2 0. So- 
lutions for which the solvent is not specified are aqueous 
solutions. 

(12) In making up solutions of definite percentage, it is 
understood that x g substance is dissolved in H 2 and diluted 
to 100 mL. Although not theoretically correct, this convention 
will not result in any appreciable error in any methods given 
in this book. 

(13) Chromic acid cleaning solution is prepared by (7) add- 
ing 1 L H 2 S0 4 to approx. 35 mL saturated aqueous Na 2 Cr 2 7 
solution; or (2) adding 2220 mL (9 lb) H 2 S0 4 to approx. 25 
mL saturated aqueous Cr0 3 solution (170 g/100 mL). Re- 
agents may be technical high grade. Use only after first clean- 
ing by other means (e.g., detergent) and draining. Mixture is 
expensive and hazardous. Use repeatedly until it is diluted or 
has a greenish tinge. Discard carefully with copious amounts 
of H 2 0. 

(14) All calculations are based on table of international atomic 
weights. 



xv 



Apparatus 

(15) Burets, volumetric flasks, and pipets conform to the 
following U.S. Federal specifications (available from General 
Services Administration, Specification Activity 3F1, Wash- 
ington Navy Yard, Building 197, Washington/ DC 20407): 



Buret 


NNN-B-00789a 


May 19, 1965 


Flask, vol. 


NNN-F-289d 


Feb. 7, 1977 


Pipet, vol. 


NNN-P-395d 


Feb. 24, 1978 


Pipet, measuring 


NNN-P-35UC 


July 16, 1973 



See also Appendix V, "Testing of Glass Volumetric Appara- 
tus," in NIST Specification Publication 260-54, "Certification 
and Use of Acidic Potassium Dichromate Solutions as an Ul- 
traviolet Absorbance Standard SRM935" (available from NIST, 
Office of Standard Reference Materials, B3I6 Chemicals, 
Gaithersburg, MD 20899). 

(16) Standard taper (I) glass joints may be used instead of 
stoppers where the latter are specified or implied for connect- 
ing glass apparatus. 

(17) Sieve designations, unless otherwise specified, are those 
described in U.S. Federal Specification RR-S-366e, Novem- 
ber 9, 1973 (available from General Services Administration). 
Designation "100 mesh" (or other number) powder (material, 
etc.) means material ground to pass through standard sieve No. 
100 (or other number). Corresponding international standard 
and U.S. standard sieves are given in Table 1. 

(18) Term "paper" means filter paper, unless otherwise 
specified. 

(19) Term "high-speed blender" designates mixer with 4 
canted, sharp-edge, stainless steel blades rotating at the bottom 
of 4-lobe jar at 10,000-12,000 rpm, or with equivalent shear- 
ing action. Suspended solids are reduced to fine pulp by action 
of blades and by lobular container, which swirls suspended 
solids into blades. Waring Blender, or equivalent, meets these 
requirements. 

(20) "Flat-end rod" is glass rod with one end flattened by 
heating to softening in flame and pressing vertically on flat 
surface to form circular disk with flat bottom at end. 

(21) Designation and pore diameter range of fritted glass- 
ware are: extra coarse, 170-220 jxm; coarse, 40-60; medium, 
10-15; fine, 4-5.5; Jena designations and pore diameter are: 
1, 110 ujn; 2, 45; 3, 25; 4, 8. 

(22) Unless otherwise indicated, temperatures are expressed 
as degrees Centigrade. 

Standard Operations 

(23) Operations specified as "wash (rinse, extract, etc.) with 
two (three, four, etc.) 10 mL (or other volumes) portions of 
H 2 (or other solvent)" mean that the operation is to be per- 
formed with indicated volume of solvent and repeated with 
same volume of solvent until number of portions required have 
been used. 

(24) Definitions of terms used in methods involving spec- 
trophotometry are those given in JAOAC 37, 54(1954). Most 
important principles and definitions are: 

(a) More accurate instrument may be substituted for less 
accurate instrument (e.g., spectrophotometer may replace col- 
orimeter) where latter is specified in method. Wavelength 
specified in method is understood to be that of maximum ab- 
sorbance (A), unless no peak is present. 

(b) Absorbance(s) (A). — Negative logarithm to base 10 of 
ratio of transmittance (T) of sample to that of reference or 
standard material. Other names that have been used for quan- 
tity represented by this term are optical density, extinction, 
and absorbancy. 

(c) Absorptivity(ies) (a). — Absorbance per unit concentra- 



Table 1. Nominal Dimensions of Standard Test Sieves (USA 






Standard Series) 










Sieve Designation 




Nominal 


Nominal 




International 




Sieve 


Wire 




Standard 3 U.S.A. 


Opening, 


Diameter, 






(ISO) Standard 


inches 


mm 




12.5 


mm 6 V2 i 


n. & 


0.500 


2.67 




11.2 


mm 7 / i6 


in. 


0.438 


2.45 


9.5 


mm 3 /s i 


n. 


0.375 


2.27 


ao 


mm 5 / ]6 


in. 


0.312 


2.07 


6.7 


mm 0.265 in. 


0.265 


1.87 


6.3 


mm" 'a i 


n b 


0.250 


1.82 


5.6 


mm No. 


3Va 


0.223 


1.68 


4.75 


mm No. 


4 


0.187 


1.54 


4.00 


mm No. 


5 


0.157 


1.37 


3.35 


mm No. 


6 


0.132 


1.23 


2.80 


mm No. 


7 


0.111 


1.10 


2.38 


mm No. 


8 


0.0937 


1.00 


2.00 


mm No. 


10 


0.0787 


0.900 


1.70 


mm No. 


12 


0.0661 


0.810 


1.40 


mm No. 


14 


0.0555 


0.725 


1.18 


mm No. 


16 


0.0469 


0.650 


1.00 


mm No. 


18 


0.0394 


0.580 


850 


ixm c No. 


20 


0.0331 


0.510 


710 


M,m No. 


25 


0.0278 


0.450 


600 


fxm No. 


30 


0.0234 


0.390 


500 


ixm No. 


35 


0.0197 


0.340 


425 


ixm No. 


40 


0.0165 


0.290 


355 


ixm No. 


45 


0.0139 


0.247 


300 


|jim No. 


50 


0.0117 


0.215 


250 


ixm No. 


60 


0.0098 


0.180 


212 


^m No. 


70 


0.0083 


0.152 


180 


jam No. 


80 


0.0070 


0.131 


150 


fxm No. 


100 


0.0059 


0.110 


125 


fxm No. 


120 


0.0049 


0.091 


106 


ixm No. 


140 


0.0041 


0.076 


90 


|xim No. 


170 


0.0035 


0.064 


75 


jxm No. 


200 


0.0029 


0.053 


63 


ixm No. 


230 


0.0025 


0.044 


53 


M,m No. 


270 


0.0021 


0.037 





"These standard designations correspond to the values for test sieve ap- 
ertures recommended by the International Organization for Standardization, 
Geneva, Switzerland. 

fa These sieves are not in the standard series but they have been included 
because they are in common usage. 

c 1000 ixm = 1 mm. 



tion and cell length, a = A /be, where b is cm and c is g/L, 
or a - (A J be) x 1000, if c is mg/L. Other names that have 
been used for this or related quantities are extinction coeffi- 
cient, specific absorption, absorbance index, and Ej^. 

(d) Transmittance(s) (T). — Ratio of radiant power trans- 
mitted by sample to radiant power incident on sample, when 
both are measured at same spectral position and with same slit 
width. Beam is understood to be parallel radiation and incident 
at right angles to plane parallel surface of sample. If sample 
is solution, solute transmittance is quantity usually desired and 
is detected directly as ratio of transmittance of solution in cell 
to transmittance of solvent in an equal cell. Other names that 
have been used for this quantity are transmittancy and trans- 
mission. 

(e) Standardization. — Spectrophotomer may be checked for 
accuracy of wavelength scale by referring to Hg lines: 239.94, 
248.3, 253.65, 265.3, 280.4, 302.25, 313.16, 334.15, 365.43, 
404.66, 435.83, 546.07, 578.0, and 1014.0 nm. To check 
consistency of absorbance scale, prepare solution of 0.0400 g 
K 2 Cr0 4 /L 0.05N KOH and determine absorbance at following 
wavelengths in 1 cm cell: 230 nm, 0.171; 275, 0.757; 313.2, 
0.043; 375, 0.991; 400, 0.396. See NIST Spec. Pub. 378, 
"Accuracy in Spectrophotometry and Luminescence Measure- 
ments," 1973 (available from NIST, Office of Standard Ref- 
erence Materials, B316, Chem., Gaithersburg, MD 20899). 



(25) Least square treatment of data and calculation of 
regression lines.-— This technique finds the best fitting straight 
line for set of data such as standard curve. It calculates that 
straight line for which sum of squares of vertical deviations 
(usually A) of observations from the line is smaller than cor- 
responding sum of squares of deviation from any other line. 
Equation of straight line is: 

Y = a + bX 

where a is intercept at Y axis (X = 0), and b is slope of line. 
Least square estimates of constants are: 



b = - 



a = Y 



2(XXd - [<JX£Yi)/n\ 



2X1 
- bX 



axf/n 



where X = "sum of" the n individual values of indicated op- 
eration, and X and Y are the averages of the X and Y points. 
Example: To find "best" straight line relating A (Y) to con- 
centration (X): 



Observation 


Concn 


Absorbance 






No. (,-) 


K 


Ys 


x? 


XX, 


1 


80 


1.270 


6400 


101.6 


2 


60 


1.000 


3600 


60.0 


3 


40 


0.700 


1600 


28.0 


4 


30 


0.550 


900 


16.5 


5 


20 


0.250 


400 


5.0 


6 


10 


0.100 


100 


1.0 


7 





0.050 





0.0 


Totals: 










n - 7 


2X, = 240 


2Y; = 3.92 


2X? 

= 13000 


2(XXi) 
= 212.1 



X - IX; /n - 240/7 - 34.29 
Y=2Yj/n = 3.92/7 = 0.56 

212.1 - (240)(3.92)/7 77.7 

b = = = 0.0163 

13000- (240) 2 /7 4771 
a - 0.56 - 0.0163(34.29) = 0.001 



Best equation is then: 



If for sample, A 
be: 



Y = 0.00 + 0.01 63X 
: 0.82, corresponding concentration (X) would 



X = (Y - 0.00)/0.0163 
Many scientific and 



0.82/0.0163 - 50.3 



statistical calculators are prepro- 
grammed to perform this calculation. 

(26) Recovery (R) of analyte from fortified sample by a 
method of analysis. — Fraction of an analyte added to a sample 
(fortified sample) prior to analysis, which is measured (re- 
covered) by the method. When the same analytical method is 
used to analyze both the unfortified and fortified samples, cal- 
culate %R as follows: 

%R - [(C F - Cu)/C A ] X 100 

where C F = concentration of analyte measured in fortified 
sample; 
Cu = concentration of analyte measured in unfortified 

sample; 
C A — concentration of analyte added in fortified sam- 
ple. 
(Note: C A is a calculated value, not a value measured by the 
method being used.) 

Concentration of added analyte should be no less that con- 
centration of analyte in unfortified sample. Sum of concentra- 
tion of added analyte plus analyte present before fortification 



should be in same range as analyte concentration sought in 
actual samples. Addition of analyte must not cause measuring 
instrument to exceed linear dynamic range of standard curve. 
Both fortified and unfortified samples must be treated identi- 
cally during analysis to minimize experimental bias. 

(27) Common safety precautions are given in the safety 
chapter. 

Method Performance 

(28) Efforts are being made to standardize the symbols and 
associated definitions for the statistical parameters that will ac- 
company approved methods. Users of the method should con- 
sult the report of the collaborative study (reference given with 
the method) for complete details. 

Beginning with methods published in "Changes in Official 
Methods of Analysis" (1989) JAOAC 72, 188, the following 
statistical parameters are shown. Data from some studies may 
not be amenable to provide these measures of evaluation. 

Within -laboratory precision: 

s r repeatability standard deviation 
s R reproducibility standard deviation 

Among-laboratories precision: 

RSD r repeatability relative standard deviation 
RSD R reproducibility relative standard deviation 

Surplus Methods 

(29) * This symbol indicates a method which has been de- 
clared surplus. Such methods are satisfactory methods, having 
been subjected to collaborative study and review. They are 
thought not to be in current use for various reasons: The pur- 
pose for which the method was developed no longer exists; the 
product for which the method was developed is no longer mar- 
keted; the method has been replaced by other methods; etc. 
These methods retain their official status but are carried only 
by reference. Any laboratory which uses a surplus method and 
wishes the text reprinted in the next edition must so notify 
AOAC. 

Editorial Conventions 

(30) For sake of simplicity, abbreviations CI and I instead 
of Cl 2 and I 2 are used for chlorine and iodine. Similar abbre- 
viations have been used in other cases (O, N, H). The same 
abbreviation may also be used for the ion where no ambiguity 
will result. 

(31) Reagents and apparatus referenced with only a letter, 
e.g., (c), will be found in the reagent or apparatus section of 
that method. 

(32) To conserve space, most of the articles and some prep- 
ositions have been eliminated. 

Manufacturers and Suppliers 

(33) Names and addresses of manufacturers and suppliers, 
and trade names of frequently mentioned materials, are fur- 
nished below solely as a matter of identification and conve- 
nience, without implication of approval, endorsement, or cer- 
tification. The same products available from other suppliers or 
other brands from other sources may serve equally well if proper 
tests indicate their use is satisfactory. These firms when men- 
tioned in a method are given by name only (without ad- 
dresses). 



Ace Glass Inc., 1430 Northwest Blvd, Vineland, NJ 08360 
Aldrich Chemical Co., Inc., 940 W St. Paul Ave, Milwaukee, 

WI 53233 
Alltech Associates, Inc., 2051 Waukegon Rd, Deerfield, IL 

60015 



American Cyanamid Co., Agricultural Div., 1 Cyanamid Plaza, 

Wayne, NJ 07470 
(ASBC) American Society of Brewing Chemists, 3340 Pilot Knob 

Rd, St. Paul, MN 55121 
(ATCC) American Type Culture Collection, 12301 Parklawn 

Dr, Rockville, MD 20852 
Analabs Inc., 140 Water St, Norwalk, CT 06854 
Analtech Inc., 75 Blue Hen Dr, PO Box 7558, Newark, DE 

19714 
Applied Science, 2051 Waukegan Rd, Deerfield, IL 60015 
J.T. Baker, Inc., 222 Red School Ln, Phillipsburg, NJ 08865 
BBL Microbiology Systems, Div. of Becton, Dickinson & Co. , 

PO Box 243, Cockeysville, MD 21030 
Beckman Instruments, Inc., 2500 Harbor Blvd, PO Box 3100, 

Fullerton, CA 92634 
Bio-Rad Laboratories, 1414 Harbour Way South, Richmond, 

CA 94804 
Brinkmann Instruments, Inc., Cantiague Rd, Westbury, NY 

11590 
Bur dick & Jackson Laboratories, Inc., Div. of Baxter Health- 
care Corp., 1953 S Harvey St, Muskegon, Ml 49442 
Burrell Corp., 2223 Fifth Ave, Pittsburgh, PA 15219 
Calbiochem Corp., 10933 N Torrey Pines Rd, LaJolla, CA 

92037 
Carborundum Co., PO Box 337, Niagara Falls, NY 14302 
(CGW) Corning Glass Works, Laboratory Products Dept, 

Corning, NY 14830 
Curtin Matheson Scientific, Inc., 9999 Veterans Memorial Dr, 

PO Box 1546, Houston, TX 77038 
Difco Laboratories, PO Box 331058, Detroit, MI 48232-7058 
Dohrmann, Div. of Xertex Corp., 3240 Scott Blvd, Santa Clara, 

CA 95050 
Dow Chemical Co., Sample Coordinator, 9001 Bldg, PO Box 

1706, Midland, MI 48647-1706 
Dow Corning Corp., PO Box 999, Midland, MI 48686-0997 
EJ. du Pont de Nemours & Co., Inc., Instrument Products 

Div., Concord Plaza, 1007 Market St, Wilmington, DE 

19898 
Eastman Kodak Co., Eastman Organic Chemicals, 343 State 

St, Rochester, NY 14650 
Elanco Products Co., Div. of Eli Lilly & Co., Elanco Ana- 
lytical Laboratories, Lilly Corporate Center, Indianapolis, 

IN 46285 
EM Sciences, A Div. of EM Industries, 480 Democrat Rd, 

Gibbstown, NJ 08027 
Fischer & Porter Co., Lab Crest Scientific, E County Line 

Rd, Warminster, PA 18974 
Fisher Scientific Co., 1 Reagent Ln, Fair Lawn, NJ 07410 
Floridin Co., 3 Penn Center, Pittsburgh, PA 15235 
Foss Food Technology Corp., 10355 W 70th St, Eden Prairie, 

MN 55344 
Gelman Scientific Inc., 600 S Wagner Rd, Ann Arbor, MI 

48106 
Gist-Brocades USA, PO Box 241068, Charlotte, NC 28224- 

1068 
Hamilton Co., PO Box 17500, Reno, NV 89510 
Hess & Clark Laboratories, Div. of Rhodia, Inc., 7th & Or- 
ange Sts, Ashland, OH 44805 
Hewlett-Packard Co., Avondale Div., Rte 41, PO Box 900, 

Avondale, PA 19311-0900 
Hewlett-Packard Co., Mail Stop 20B3, 3000 Hanover St, Palo 

Alto, CA 94304 
Hoffmann-La Roche, Inc., 340 Kingsland St, Nutley, NJ 07110 
ICI Americas, Inc., Western Research Center, 1200 S 47th St, 

PO Box 4023, Richmond, CA 94804-0023 
ICN Pharmaceuticals, Inc., Life Sciences Group, 26201 Miles 

Rd, Cleveland, OH 44128 



(IEC) International Equipment Co., Div. of Damon, 300 Sec- 
ond Ave, Needham Heights, MA 02194 
Kimble Glass Inc., Crystal Ave, Vineland, NJ 08360 
Kontes Glass Co., Spruce St, Vineland, NJ 08360 
Kopp Glass Co., 2108 Palmer St, Swissvale, PA 15218 
Labconco Corp., 88 1 1 Prospect Ave, Kansas City, MO 64132 
Lurex Scientific, 1298 North West Blvd, PO Box 2420, Vine- 
land, NJ 08360 
Mallinckrodt Chemicals Works, Science Products Div., 675 

McDonnell Blvd, PO Box 5840, St. Louis, MO 63134 
Manville Filtration & Minerals, PO Box 519, Lompoc, CA 

93438 
Matheson Scientific, Inc., see Curtin Matheson Scientific, Inc. 
E. Merck, Frankfurter Str 250, Postfach 4119, D6100 Darm- 
stadt, West Germany 
Merck & Co., Inc., Chemical Div., PO Box 2000, Rahway, 

NJ 07065 
Mettler Instrument Corp., PO Box 71, Hightstown, NJ 08520 
Millipore Corp., Ashby Rd, Bedford MA 01730 
Mobay Corp., Agricultural Chemicals Div., Hawthorne Rd, 

PO Box 4913, Kansas City, MO 64120-0013 
Monsanto Chemical Co., 800 N Lindberg Blvd, St. Louis, 

MO 63167 
(NBS) National Bureau of Standards, see NIST 
(NF) National Formulary, see USP 
New York Laboratory Supply Co., 510 Hempstead Tnpk, West 

Hempstead, NY 11552 
(NIST) National Institute of Standards and Technology, Gaith- 

ersburg, MD 20899 
Orion Research Inc., 529 Main St, Boston, MA 02149 
Perkin-Elmer Corp., 761 Main Ave, Mail Station 256, Nor- 
walk, CT 06859-0256 
Pierce Chemical Co., PO Box 117, Rockford, IL 61105 
Rheodyne Inc., PO Box 996, Cotati, CA 94928 
Rohm & Haas Co., Independence Mall West, Philadelphia, 

PA 19105 
Salsbury Laboratories, Charles City, I A 50616 
Sargent-Welch Scientific Co., 7300 N Linder Ave, PO Box 

1026, Skokie, IL 60077 
(S&S) Sleicher & Schuell, Inc., 10 Optical Ave, Keene, NH 

03431 
(SEPCO) Scientific Equipment Products Co., Div. of JAG In- 
dustries, Inc., 2201 Aisquith St, Baltimore, MD 21218 
Scientific Products Inc., Div. of Baxter Healthcare Corp., 1430 

Waukegan Rd, McGaw Park, IL 60085-6787 
Searle Analytic, Inc., 2000 Nuclear Dr, Des Plaines, IL 60018 
Shell Chemical Co., 1 Shell Plaza, Houston, TX 77002 
Sigma Chemical Co., PO Box 14508, St. Louis, MO 63178 
G. Frederick Smith Chemical Co., PO Box 23214, Columbus, 

OH 43223 
Supelco, Inc., Supelco Park, Bellefonte, PA 16823-0048 
Technicon Instruments Corp., 511 Benedict Ave, Tarry town, 

NY 10591 
Thomas Scientific, 99 High Hill Rd, 1-295, PO Box 99, Swe- 

desboro, NJ 08085 
Tracor Instruments, Austin, Inc., 6500 Tracor Ln, Bldg 27-7, 

Austin, TX 78726-2100 
UVP, Inc., 5100 Walnut Grove Ave, PO Box 1501, San Ga- 
briel, CA 91778-1501 
Union Carbide Corp., Old Ridgebury Rd, Danbury, CT 06817 
Uniroyal Chemical, Elm St, Naugatuck, CT 06770 
The Upjohn Co., 7000 Portage Rd, Kalamazoo, MI 49001 
(USP) United States Pharmacopeia!. Convention, Inc., 12601 

Twinbrook Pkwy, Rockville, MD 20852 
Varian Instrument Group, 505 Julie Rivers Rd, Su gad and, TX 

77478 
VWR Scientific, PO Box 7900, San Francisco, CA 94120 



Waters Associates, Inc., 34 Maple St, Milford, MA 01757 
Whatman, Inc., 9 Bridewell PI, Clifton, NJ 07014 

Abbreviations 

(34) The following abbreviations, many of which conform 
with those of Chemical Abstracts, are used. In general, prin- 
ciple governing use of periods after abbreviations is that period 
is used where final letter of abbreviation is not the same as 
final letter of word it represents. Periods are not used with 
units, except inch(es) and gallon(s). Hour(s), second(s), inch(es), 
and foot(feet) appear as hr or h, sec or s, " or in., and ' or ft, 
because of new abbreviations adopted in recent methods. 

Abbreviation Word 

a absorptivity(ies) 

A absorbance(s) thruout (not restricted to for- 
mulas); not absorption. A' is used for std; Ao 
for blank; 3 digit subscript numerals usually 
denote wavelengths in nm 

AA atomic absorption 

Ac CH 3 CO- (acetyl, not acetate) 

ACS American Chemical Society 

addn addition 

addnl additional 

ale. alcoholic (not alcohol) 

alk. alkaline (not alkali) 

alky alkalinity 

amp ampere(s) 

amt amount 

anal. analytical(ly) 

anhyd. anhydrous 

AOCS American Oil Chemists' Society 

APHA American Public Health Association 

app. apparatus 

approx. approximate(Iy) 

aq. aqueous 

ASTM American Society for Testing and Minerals 

arm. atmosphere, atmospheric 

av. average (except as verb) 

Be. degree Baume 

bp boiling point 

Bu butyl 

C degrees Celsius (Centigrade) 

ca about, approximately 

calc. calculate 

calcd calculated 

calcg calculating 

calcn calculation 

Cat. No. Catalog Number 

centrf. centrifuge 

centrfd centrifuged 

centrfg centrifuging 

Chap. Chapter 

chem. chemical (ly) 

chromatgc chromatographic 

chromatgd chrornatographed 

chromatgy chromatography 

Ci curie(s) 

CI Colour Index 

CIPAC Collaborative International Pesticides Analyti- 
cal Council 

cm centimeter(s) 

compd compound 

com. commercial(ly) 

cone. concentrate (as verb or noun) 

coned concentrated 

concg concentrating 



Abbreviation 


Word 


concn 


concentration 


const 


constant 


contg 


containing 


cP 


centipoise 


cpm 


counts per minute 


cryst. 


crystalline (not crystallize) 


crystd 


crystallized 


crystg 


crystallizing 


crystn 


crystallization 


cu in. 


cubic inch(es) 


dc 


direct current 


det. 


determine 


detd 


determined 


detg 


determining 


detn 


determination 


diam. 


diameter 


diat. earth 


diatomaceous earth 


dil. 


dilute 


dild 


diluted 


dilg 


diluting 


diln 


dilution 


distd 


distilled 


distg 


distilling 


distn 


distillation 


DMF 


N , N-di methy If orm amide 


DMSO 


dimethyl sulfoxide 


EDTA 


ethylenedinitrilotetraacetic acid (or -tetraace- 




tate) 


e.g. 


for example 


elec. 


electric(al) 


equiv. 


equivalent 


est. 


estimate 


estd 


estimated 


estg 


estimating 


estn 


estimation 


Et 


ethyl 


EtOH 


ethanol (the chemical entity C 2 H 5 OH) 


evap. 


evaporate 


evapd 


evaporated 


evapg 


evaporating 


evapn 


evaporation 


ext 


extract 


extd 


extracted 


extg 


extracting 


extn 


extraction 


F 


degrees Fahrenheit (°C - (5/9) X (°F - 32)) 


FAO 


Food and Agriculture Organization 


Fig. 


Figure (illustration) 


fl oz 


fluid ounce(s) (29.57 mL) 


fp 


freezing point 


ft 


foot (30.48 cm) 


g 


gram(s) 


g 


gravity (in centrfg) 


gal. 


gallon(s) (3.785 L) 


GC 


gas chromatography 


gr- 


grain(s) 


g-s 


glass- stoppered 


HCHO 


formaldehyde 


HOAc 


acetic acid (not HAc) 


h or hr 


hour(s) 


ht 


height 


id 


inner diameter (or dimension) 


in. 


inch(es) (2.54 cm) 


inorg. 


inorganic 


insol. 


insoluble 



XIX 



Abbreviation 


Word 


Abbreviation 


Word 




IR 


infrared 


pptd 


precipitated 




ISO 


Internationa] Organization for Standardization 


PPtg 


precipitating 




JAOAC 


Journal of the Association of Official Analyt- 


pptn 


precipitation 






ical Chemists (after 1965) 


Pr 


propyl 






Journal of the Association of Official Agricul- 


prep. 


prepare 






tural Chemists (before 1966) 


prepd 


prepared 


kg 


kilogram(s) 


prepg 


preparing 


L 


liter(s) 


prepn 


preparation 


LC 


liquid chromatography 


psi 


pounds per square inch (absolute) 


lb 


pound(s) (453.6 g) 


psig 


pounds per square inch gage (atmospheric 


liq. 


liquid 




pressure = 0) 


in 


meter(s); milli — as prefix 


Pt 


pint(s) (473 mL) 


m 


molal 


QAC 


quaternary ammonium compound 


M 


molar (as applied to concn), not molal 


qt 


quart(s) (946 mL) 


ma 


milliampere (cf amp) 


qual. 


qualitative(ly) 


mag. 


magnetic(ally) 


quant. 


quantitative(ly) 


manuf. 


manufacturer 


® 


Trademark name — (Registered) 


max. 


maximum 


Rr 


distance spot moved/distance solv. moved 


mech . 


mechanical(ly) 




(TLC) 


Me 


methyl 


r-b 


round-bottom (flask) 


MeOH 


methyl alcohol 


ref. 


reference 


mg 


milligram(s) 


resp. 


respectively 


min 


minute(s) 


rpm 


revolutions per minute 


min. 


minimum 


sat. 


saturate 


mixt. 


mixture 


satd 


saturated 


ml 


milliliters) 


satg 


saturating 


mm 


millimeter(s) 


satn 


saturation 


mp 


melting point 


-SCN 


thiocyanate 


mp. 


millimicron (10~ 6 mm); use nanometer (nm) 


SDF 


special denatured formula (applied to alcohol) 




(1(T 9 m) 


s or sec 


second(s) 


mv 


millivolt 


sep. 


separate (ly) 


MW 


molecular weight 


sepd 


separated 


TV 


normal (as applied to concn); in equations, 


sepg 


separating 




normality of titrating reagent 


sepn 


separation 


N 


Newton (10 5 dynes) 


sol. 


soluble 


n 


refractive index 


soln 


solution 


neg. 


negative 


solv. 


solvent 


neut. 


neutral 


sp gr 


specific gravity (apparent density) 


neutze 


neutralize 


spectrophtr 


spectrophotometer 


neutzd 


neutralized 


spectrophtric 


spectrophotometric(ally) 


neutzg 


neutralizing 


sq 


square 


neutzn 


neutralization 


SRM 


Standard Reference Material of National Insti- 


NF 


National Formulary 




tute of Standards and Technology 


NFPA 


National Food Processors Association 


std 


standard 


HIST 


National Institute of Standards and Technology 


std dev. 


standard deviation 


ng 


nanogram (10~ 9 g) 


stdzd 


standardized 


nm 


nanometer (10~ 9 m); formerly mp, 


stdze 


standardize 


No. 


number 


stdzg 


standardizing 


-OAc 


acetate (cf Ac) 


stdzn 


standardization 


-OCN 


cyanate 


T 


transmittance 


od 


outer diameter (or dimension) 


tech. 


technical 


org. 


organic 


temp. 


temperature 


oxidn 


oxidation 


titr. 


titrate 


oz 


ounce(s) (28.35 g) 


titrd 


titrated 


p 


pico (10~ 12 ) as prefix 


titrg 


titrating 


Pa 


Pascal (1 Newton/m 2 ; 9.87 X 10" 6 atm.; 7.5 


titrn 


titration 




X 10" 3 mm Hg (torr); 1.45 X 1(T 4 psi) 


TLC 


thin layer chromatography 


par. 


paragraph(s) 


U 


unit 


pet ether 


petroleum ether 


USDA 


United States Department of Agriculture 


phthln 


phenolphthalein 


USP 


United States Pharmacopeia 


pos. 


positive 


uv 


ultraviolet 


powd 


powdered (as adjective) 


v 


volt(s) 


ppb 


parts per billion (1/10 9 ) 


v/v 


both components measured by vol. 


ppm 


parts per million (1/10 6 ) 


vac. 


vacuum 


ppt 


precipitate 


vol. 

XX 


volume; also volumetric when used with flask 





Abbreviation 


Word 


Abbreviation 


w/w 


both components measured by wt 


1 


WHO 


World Health Organization 


% 


wt 


weight 


> 


M- 


micron (0.001 mm); use micrometer (jim) (10~ 6 






m) 


< 


M£ 


microgram(s) (10 6 g) 


^ 


jjlL 


microliter(s) (10~ 6 L) 




(am 


micrometer(s) (10~ 6 m); formerly jjl 


> 


A 


difference (e.g., &A - (A - A')) 




' 


foot (feet) (T = 30.48 cm) 


I 


" 


inch(es) (1" - 2.54 cm) 


J 



Word 

per 

percent (parts per 100); percentage 

more than; greater than; above; exceeds (use 

with numbers only) 
less than; under; below (use with numbers only) 
not more than; not greater than; equal to or less 

than 
not less than; equal to or greater than; equal to 

or more than; at least 
standard taper 
standard spherical joint 



Collaborative Study Procedures of the Association 
of Official Analytical Chemists 



The Association of Official Analytical Chemists (AOAC) is 
a unique, nonprofit scientific organization whose primary pur- 
pose is to serve the needs of academia, government regulatory 
and research agencies, and industry for analytical methods for 
compliance, quality control, and research purposes. The goal 
of the Association is to provide methods which will perform 
with the necessary accuracy and precision under usual labo- 
ratory conditions (/). Since its formation in 1884, AOAC has 
provided a mechanism to select methods of analysis from pub- 
lished literature or develop new methods, collaboratively test 
them through interlaboratory studies, approve them, and pub- 
lish the approved methods for a wide variety of materials re- 
lating to foods, drugs, cosmetics, pesticides, feeds, fertilizers, 
forensic science, and products affecting the public health and 
welfare. Its membership is composed of scientists from gov- 
ernment, academia, and industry laboratories in many coun- 
tries who work within AOAC's established procedures as re- 
searchers, methods collaborators, and committee members. 

AOAC has more than 100 years of experience in utilizing 
the collaborative study as a means of determining the reli- 
ability of analytical methods for general purposes and, espe- 
cially, for regulatory purposes. In fact, AOAC's major con- 
tribution to analytical science has been to bring the collaborative 
study technique for the validation of analytical methods to a 
high degree of perfection . In such a study, laboratories analyze 
identical sample sets which cover the range of applicability of 
a method previously selected as being useful and practical. 
The purpose of the study is to establish the characteristics of 
the method with respect to accuracy, precision, sensitivity, 
range, specificity, limit of detection, .limit of reliable mea- 
surement, selectivity, practicality, and similar attributes, as re- 
quired. 



ORGANIZATION AND PROCEDURES FOR AOAC 
COLLABORATIVE STUDIES 

The collaborative study is organized and directed by an an- 
alyst designated as the Associate Referee for the specific sub- 
ject under investigation. Currently, some 700 Associate Ref- 
erees appointed by the Association are responsible for as many 
topics. An Associate Referee is selected for his or her knowl- 
edge, interest, and experience in the subject matter field. The 
Associate Referee operates under the scientific guidance and 
support of a General Referee, who is in turn responsible for a 
product area. The Associate Referee reviews the literature and 
selects one or two of the appropriate analytical methods avail- 
able, modifying them as needed. Alternatively, he or she may 
develop or adapt a method used in his or her laboratory for 
the analyte and matrix under study, testing it thoroughly before 
designing a collaborative study. The General Referee is kept 
informed of such preliminary studies. 

The samples analyzed in a collaborative study are normally 
prepared and distributed to the participants by the Associate 



Referee. The Association follows the "Guidelines for Collab- 
orative Study Procedure to Validate Characteristics of a Method 
of Analysis," as accepted by IUPAC and adopted by AOAC 
(see p. 673) for the number of participating laboratories and 
number of materials. 

Laboratories with at least some experience in the general 
subject matter are selected as collaborators. Because the ob- 
jective of the study is to evaluate the method, as contrasted to 
evaluating the analyst (2), all analysts are instructed to follow 
the method exactly as written even though they may not concur 
with the Associate Referee's selection among possible alter- 
natives. The content of the analyte in the samples is unknown 
to the participants. 

All individual results obtained by the collaborators are re- 
ported to the Associate Referee, who compiles and evaluates 
them. Since statistical treatment of the data is considered es- 
sential in a rigorous evaluation of the method for accuracy, 
precision, sensitivity, and specificity, it is now required for all 
studies. The Association considers this of such importance that 
it provides statistical assistance in all cases where it is other- 
wise unavailable to the Associate Referee. A statistical manual 
(3) is also provided. 

The Associate Referee makes the initial judgment on the 
performance of the method. If he or she recommends ap- 
proval, it passes to the General Referee, the appropriate Meth- 
ods Committee, and then to the chairman of the Official Meth- 
ods Board. If all parties recommend approval, the method 
receives interim official first action approval. The method is 
then presented at the Association's annual business meeting 
for vote for adoption as official by the membership, 

Approved methods and supporting data are published in the 
Journal of the Association of Official Analytical Chemists. They 
are subject to scrutiny and general testing by other analysts for 
2 years before final adoption. They may be modified and re- 
studied collaboratively as needed, should feedback from gen- 
eral use reveal flaws in the method or in its written set of 
directions. Approved methods are included in the Associa- 
tion's Official Methods of Analysis, the compendium of all 
adopted methods, which is updated every 5 years. 

The preceding summary of AOAC's modus operandi rec- 
ognizes the need for healthy skepticism toward results ob- 
tained by analytical methods which have not undergone such 
rigorous scrutiny and interlaboratory testing of their accuracy, 
precision, specificity, and practicality. 



SELECTION OF METHODS FOR STUDY 

A certain degree of variability is associated with all mea- 
surements. Much of the research on analytical chemistry is an 
attempt to minimize that variability. But there are many dif- 
ferent types of variability in analytical work. We often find 
that when we attempt to minimize one kind, we must neces- 
sarily permit expansion in another kind. In practical analytical 



XXII 



chemistry, the problem often comes down to which variability 
is to be minimized. 

Some examples of this point may be helpful. In atomic weight 
determination, everything — especially practicality — is sacri- 
ficed for accuracy. A high degree of accuracy and practicality 
is required in the assay of precious metals, but the fire assay 
used is generally applicable to little else besides metals and 
minerals. In clinical chemistry, within-laboratory precision 
(repeatability) is critical, and often is of greater interest to clin- 
ical laboratories than absolute accuracy or agreement with the 
values of other laboratories (reproducibility). In drug analysis, 
a high degree of accuracy is required in the therapeutic range 
because the analytical values determining the identity, strength, 
quality, and purity of pharmaceutical preparations, as laid down 
in pharmacopoeial specifications, are directly related to clin- 
ical value. With poly nuclear hydrocarbons, specificity is im- 
portant, since some of these compounds are carcinogenic while 
others are not. In applying the famous Delaney clause of the 
United States Federal Food, Drug, and Cosmetic Act, all at- 
tributes of the analytical methods are secondary to the detec- 
tion of extremely small concentrations (detectability), or to ex- 
hibiting a high degree of response for small changes in 
concentration (sensitivity) . 

There is a very special case involving accuracy, where the 
"true value" is determined by the method of analysis. Many 
legal specifications and standards for food and agricultural 
products define ill-defined components such as moisture, fat, 
protein, and crude fiber in terms of reference methods. There- 
fore, the precision of these methods becomes the limiting fac- 
tor for their performance. In fact, most analyses involved in 
commercial transactions require primarily that the buyer and 
seller agree on the same value (analytically and economically), 
regardless of where it stands on an absolute scale. 

The point of these examples is that although methods of 
analysis are characterized by a number of attributes — accu- 
racy, precision, specificity, sensitivity, detectability, depend- 
ability, and practicality — no method is so flawless that all these 
qualities can be maximized simultaneously. For any particular 
analysis, the analyst must determine, on the basis of the pur- 
pose of the analysis, which attributes are essential and which 
may be compromised. 

Unfortunately, the literature is replete with examples indi- 
cating that an individual analyst, and especially the originator 
of a method of analysis, is not an unbiased judge of the relative 
merits of the methods of analysis which he or she develops 
and uses. In our experience, the collaborative study provides 
impartial data on the suitability of the method. The data, in 
many cases, speak for themselves. 

The collaborative study, or ring test or round robin test, as 
it is called in other organizations, provides the basic infor- 
mation on the performance of analytical methods. The extent 
of the information will depend on the number of samples pro- 
vided, the number of analyses performed, and the number of 
laboratories participating. The data should be unbiased be- 
cause the composition of the samples is known only to the 
administrator of the study. Some of the requirements of the 
study and their relationship to the characteristics and attributes 
of the method are as follows: 

(1) Accuracy. Samples must be of defined composition (by 
spiking, by formulation, or by analytical consensus). 

(2) Specificity. Samples should contain related analytes. 

(3) Sensitivity. Samples should differ from each other or from 
negative samples by a known amount. 

(4) Applicability. Samples should include the concentration 
range and matrix components of interest. 

(5) Blanks. Samples should include different matrices with 
"none" of the component of interest. 



(6) Precision. Instructions should request replicate analyses 
by the same or different analysts in the same laboratory, pref- 
erably on different days. By far a better procedure is to include 
"blind" (unknown to the analyst) replicate samples in the se- 
ries. 

(7) Practicality. Instructions should request information as 
to the actual and elapsed time required for the analyses; the 
availability of reagents, equipment, and standards; and any 
necessary substitutions. When practice samples are included, 
the number of analyses required to achieve the stated recovery 
and repeatability should be reported. 



PROCEDURAL DETAILS OF COLLABORATIVE STUDY 

As numerous beginners in this field have discovered, much 
preliminary work must be done before sending out samples: 

(1) The method must be chosen and demonstrated to apply 
to the matrices and concentrations of interest. 

(2) The critical variables in the method should have been 
determined and the need for their control must be emphasized 
[a ruggedness test (4) is useful for this purpose]. 

(3) The method should be written in detail by the Associate 
Referee and tested by an analyst not previously connected with 
its development. 

(4) Unusual standards, reagents, and equipment must be 
available from usual commercial sources of supply, or suffi- 
cient quantities must be prepared or obtained to furnish to the 
participants. 

(5) The samples must be identical and homogeneous so that 
the analytical sample error is only a negligible fraction of the 
expected analytical error. 

(6) A sufficient number of samples must be prepared to cover 
typical matrices and the concentration range of interest (tol- 
erance, maximum or minimum specifications, likely levels of 
occurrence, etc.). 

(7) A minimum of 8 laboratories and sufficient samples must 
be included to provide a minimum of 40 data points. Addi- 
tional laboratories and samples are recommended. 

(8) Samples must be stable and capable of surviving the ri- 
gors of commercial transportation. 

(9) Reserve samples should be prepared and preserved to 
replace lost samples and to permit reanalysis of samples con- 
sidered as outliers to attempt to discover the cause of abnormal 
results. 

(10) The instructions must be clear. They should be re- 
viewed by someone not connected with the study to uncover 
potential misunderstandings and ambiguities. 

(11) If the analyte is subject to change (e.g., bacterial lev- 
els, nitroglycerin tablets), provision must be made for all par- 
ticipants to begin the analysis at the same time. 

(12) Practice samples of a known and declared composition 
should be furnished with instructions not to analyze the un- 
knowns until a specified degree of recovery and repeatability 
(or other attribute) has been achieved. 

(13) Provision should be made when necessary for submis- 
sion of standard curves, tracings of recorder charts, or pho- 
tographs of thinlayer plates in order to assist in determining 
possible causes of error. 



OTHER TYPES OF SNTERLABORATORY STUDIES 

This type of collaborative study, which is designed to de- 
termine the characteristics of a method, must be carefully dis- 



tinguished from other types of inter] aboratory studies which 
by design or through ignorance provide other kinds of infor- 
mation. The most important types of other studies are: 

(1) Those studies which require the collaborators to inves- 
tigate the variability of parts of methods or applicability to 
different types of samples. (An interl aboratory study is usually 
an inefficient way of obtaining this type of information.) 

(2) Those studies which permit an analyst to use any method 
desired. Such studies invariably produce such a wide scatter 
of results that the data are of little value for evaluation of meth- 
ods. They may be useful in selecting a method from a number 
of apparently equivalent methods, provided the purpose is em- 
phasized beforehand and the participants provide a description 
of the method used in order to permit a correlation of the de- 
tails of the methods with apparent biases and variabilities. 

(3) Those studies which are used for quality control pur- 
poses, whose participants are not permitted sufficient time to 
gain familiarity with the method, or who permit deviations to 
enter into the performance of the analyses on the grounds that 
the deviation is obviously an improvement which could not 
possibly affect the results of the analysis, or who claim to have 
a superior method. 

The following definitions were agreed on as part of the 
guidelines for collaboration between AOAC and the Collab- 
orative International Pesticides Analytical Council Ltd. (CIPAC) 
(5). 

Collaborative study. An analytical study involving a number 
of laboratories analyzing the same sample(s) by the same 
method(s) for the purpose of validating the performance of the 
method(s). 

Preliminary interlaboratory study. An analytical study in 
which two or more laboratories evaluate a method to determine 
if it is ready for a collaborative study. 

Laboratory performance check. The analysis of very care- 
fully prepared and homogeneous samples, normally of known 
active ingredient content, to establish or verify the perfor- 
mance of a laboratory or analyst. 



SUMMARY 

The collaborative study is an experiment designed to eval- 
uate the performance of a method of analysis through the anal- 
ysis of a number of identical samples by a number of different 
laboratories. With proper design, it provides an unbiased eval- 
uation of the performance of a method in the hands of those 
analysts who will use it. A collaborative study must be distin- 
guished from those studies designed to choose a method or to 
determine laboratory or analyst performance. 



REFERENCES 

(i) Handbook for AOAC Members, AOAC, Arlington, YA 
(1989). 

(2) H. Egan, "Methods of Analysis; An Analysis of Meth- 
ods," J. Assoc. Off. Anal. Chem., 60, 260-267 (1977). 

(3) W. J. Youden and E. H. Steiner, "Statistical Manual of 
the AOAC: Statistical Techniques for Collaborative Tests. 
Planning and Analysis of Results of Collaborative Tests," 
AOAC, Arlington, VA (1975). 

(4) W. J. Youden, "The Collaborative Test," J. Assoc. Off. 
Agric. Chem., 46, 55-62 (1963). 

(5) "Guidelines for Collaboration Between the Association of 
Official Analytical Chemists (AOAC) and the Collabora- 
tive International Pesticides Analytical Council Ltd. (CI- 
PAC)," J. Assoc. Off. Anal. Chem., 57, 447-449 (1974). 



BIBLIOGRAPHY 

D. Banes, "The Collaborative Study as a Scientific Concept," 
7. Assoc. Off. Anal. Chem., 52, 203-206 (1969). 

W. Horwitz, "Problems of Sampling and Analytical Meth- 
ods," J. Assoc. Off. Anal. Chem., 59, 1197-1203 (1976). 



Reprinted with permission from: Analytical Chemistry (March 1978) 50, 337A-340A, with minor 
updating revisions. Published 1978 American Chemical Society 



1. Agricultural Lsrr laterials 

Frank J. Johnson, Associate Chapter Editor 

National Fertilizer Development Center, Tennessee Valley Authority 



924.01 Sampling of Liming Materials 

Procedure 

(Caution: See safety note on calcium oxide.) 

Take sample representative of lot or shipment. Avoid dis- 
proportionate amt of surface or any modified or damaged zone. 

(a) Burnt or lump lime, in bulk. — Collect composite sample 
of ^10 shovelfuls/car, with proportionate amts from smaller 
lots, taking each shovelful from different part of lot or ship- 
ment. Immediately crush to pass 5 cm (2") diam. circular 
opening, mix thoroly and rapidly, reduce composite to ca 2 
kg (5 lb) sample by riffling or quartering, and place in labeled, 
dry, air-tight container. 

(b) Hydrated lime and ground burnt lime, in bags. — Select 
10 bags from different parts of each lot or shipment of <20 
tons and 1 addnl bag for each addnl 5 tons. Use sampling tube 
to withdraw top to bottom core from each bag selected. Com- 
bine cores, mix thoroly and rapidly, reduce composite to ca 1 
kg (2 lb) by riffling or quartering, and place in dry, air-tight 
container. 

(c) Ground limestone and ground marl, in bags. — Proceed 
as in (b). 

(d) Ground limestone, ground burnt lime, ground marl, and 
slag, in bulk. — Use slotted sampling tube to withdraw samples 
to full sampler depth from 10 points in lot or shipment. Pro- 
ceed as in (b), beginning "Combine cores, ..." 

Refs.: JAOAC 7, 252(1924); 48, 95(1965). 

CAS-1317-65-3 (limestone) 



924.02 Mechanical Analysis of 

Liming Materials 
Procedure 

(Caution: See safety note on calcium oxide.) 

If entire sample is not to be dried, obtain lesser portions by 
riffling or quartering. Dry at 1 10° to const wt and cool to room 
temp. 

Obtain 90-150 g dry sample by riffling or quartering. Break 
any agglomerates formed during drying by rolling dry sample 
with hard rubber roller on hard rubber mat, wet sieving, or by 
equally effective means that does not result in crushing the 
limestone. 

Wet sieving. — Place 100 g sample on No. 200 sieve and 
wash with moderate stream of tap H 2 at max. gage pressure 
of 0.28 kg/sq cm (4 Jb/sq in.) until H 2 passing sieve is clear, 
with care to avoid loss of sample by splashing. Dry material 
remaining on sieve at 105° and transfer to No. 100 sieve in 
series with No. 200 sieve of same diam. and depth. Shake 8 
min in mech. shaker. (If wet sieving is used to break agglom- 
erates, do wet sieving on sieve having smallest opening to be 
used in final testing. After drying, transfer to sieves to be used 
in final testing. If only 1 sieve is to be used, do not transfer.) 
Quant, transfer weighed sample to 8" diam. std sieve or set of 
sieves (e.g., Nos. 10, 20, 40, 60, 80, and 100 or other ap- 
propriate combination). 



Sieve by lateral and vertical motion accompanied by jarring 
action. Continue >5 min or until addnl 3 min of sieving time 
fails to change results of any sieve fraction by 0.5% of total 
sample wt. Do not overload any sieve when assaying closely 
sized materials. 

Det. wt of each sieve fraction and report as % of total sam- 
ple wt. 

Refs.: JAOAC 7, 252(1924); 55, 539(1972); 48, 95(1965); 52, 
322(1969). 



924.03 



Liming Materials 

Preparation of Sample 

Procedure 



Reduce dried sample, 924.02, to amt sufficient for analysis 
and grind >225 g (0.5 lb) reduced sample in mortar, ball mill, 
or other mech. app. to pass No. 60 sieve. Mix thoroly, and 
store in air-tight container. 

Refs.: JAOAC 7, 252(1924); 48, 95(1965). 



955.01 Neutralizing Value 

for Liming Materials 
Final Action 

(Uncorrected for sulfide content) 



A. Reagents 

(a) Sodium hydroxide std soln. 



0.25/V. Prep, and stdze as 

-0.5/V. Stdze against (a), 



in 936.16 

(b) Hydrochloric acid std soln 
using phthln. 

B. Indicator Titrimetric Method 

Place 0.5 g burnt or hydrated lime (1 g ground limestone or 
ground marl), prepd as in 924.03, in 250 mL erlenmeyer; add 
50 mL HC1 std soln and boil gently 5 min. Cool, and titr. 
excess acid with NaOH std soln, using phthln. For burnt and 
hydrated lime, report as % CaO; for limestone and marl, report 
as % CaC0 3 equivalence. 

% CaC0 3 equivalence of sample 

- 2.5 x (mL HC1 - mL NaOH/2) 
% CaO equivalence = 2.8 X (mL HCl - mL NaOH/2) 

C. Potentiometric Titration Method 

(Applicable to liming materials contg large amt of Fe ' 2 or col- 
oring matter, but not to silicate materials) 

Proceed as in 955.01B thru "Cool, ..." Transfer to 250 

mL beaker and insert glass and calomel electrodes of pH me- 
ter, buret contg 0.25N NaOH, and mech. stirrer. Stir at mod- 
erate speed to avoid splash. Deliver NaOH rapidly to pH 5, 
then dropwise until soln attains pH 7 and remains const 1 min 
while stirring. (If end point is passed, add, from 1 mL Mohr 
pipet, just enough 0.5iV HC1 to bring pH to <7, and back-titr. 
slowly to pH 7.) Add mL of excess acid, if used, to initial 50 



Agricultural Liming Materials 



AOAC Official Methods of Analysis (1990) 



mL in calcg. Report as % CaC0 3 or CaO equivalence as in 
955.01B 

Ref.: JAOAC 38, 240(1955). 

D. Approximate Proportions of Calcium and Magnesium in 
Magnesic Limestone 

Slightly acidify titrd soln, 955.01B or C, transfer to 250 mL 
vol. flask, and dil. to vol. Det. Ca in 50 mL aliquot as in 
927.02, beginning ". . . dil. to ca 100 mL . . ." Subtract its 
CaC0 3 equivalence from total CaC0 3 equivalence, 955. 01B 
or C, and assign difference as CaC0 3 equivalence of the Mg 
content of the limestone. 

CAS-7440-70-2 (calcium) 
CAS-131 7-65-3 (limestone) 
CAS-7439-95-4 (magnesium) 



928.01 Caustic Vafue for Liming Materials 
Titrimetric Method 
Final Action 1965 

A. Apparatus (Figure 928.01) 

Use 500 mL Pyrex erlenmeyer, A, and fritted glass filter 
(Corning Glass Works No. 39535, 30F), F. Connect filter to 
siphon tube B with thick- wall rubber tubing. Use receiving 
flasks M and TV calibrated to deliver 50 and 100 mL, resp. S 
is suction flask. 

B. Determination 

Transfer portion of sample, 924.03, to weighing bottle and 
det. wt bottle and contents in atm. of min. moisture and C0 2 
content. With polished, narrow-point spatula calibrated to hold 
ca 1.5 g, withdraw sample to be used and det. exact wt by 
difference. Insert sample directly into dry flask, A, fitted with 
tight rubber stopper. 



Prep, sucrose soln immediately before use by placing 25 g 
granulated sucrose in measuring flask calibrated to deliver 500 
mL. Dissolve sucrose with cold C0 2 -free H 2 and dil. to vol. 
Holding both erlenmeyer contg sample and flask contg sucrose 
soln in slightly inclined position, insert neck of sucrose soln 
flask short distance into erlenmeyer, and carefully transfer su- 
crose soln with synchronized rotary motion of both flasks to 
prevent granulation of lime. Stopper erlenmeyer securely, ag- 
itate, and add, if desired, some clean dry beads. Completely 
dissolve uncoated caustic lime by six 1 min agitations at 2 or 
3 min intervals. Invert flask to trap any solid particles between 
stopper and neck and crush by carefully twisting stopper. Let 
stand 15 min and filter as follows: 

Connect filter cone F with siphon B and close stopcock D. 
Connect receiving flasks, apply suction, and quickly connect 
erlenmeyer A contg lime soln with stopper E. Open stopcock 
C and filter 25-50 mL soln. Close C and open D to release 
suction. Remove M and replace with similar dry flask. Close 
D, open C, and continue filtration until both M and TV are filled 
at least to marks. To disconnect system, close stopcock C, and 
gently press down outlet of flask M and then outlet of flask 
N, to remove any excess liq. above marks. Let intermediate 
connection empty, open stopcock D, and remove M and N. 
Titr. first 50 mL, or pilot aliquot, of filtered soln with 0.5/V 
HO, using phthln. To covered 200 mL beaker add twice vol. 
0.57V acid required for this titrn, add second (100 mL) aliquot 
of filtered soln to this acid and phthln, and complete titrn, 

Calc. caustic value of sample: X - 1V/W 

where X = % active CaO; V = mL 0.5VV acid used/ 100 mL 
lime soln; W = g sample 

Refs.: Ind. Eng. Chem. 20, 312(1928). JAOAC 11, 152(1928); 
12, 146(1929). 

CAS- 1305-78-8 (calcium oxide) 




FIG. 928.01— Apparatus for automatic filtration and measurement of lime solutions 



AOAC Official Methods of Analysis (1 990) 



Slags 



955.02 Carbon Dioxide in Liming Materials 
Knorr Alkalimeter Method 
Final Action 1965 

A. Apparatus and Reagents 

Knorr alkalimeter with C0 2 absorption train. — Fill guard 
tube of alkalimeter with Ascarite. Connect upper end of con- 
denser to absorption train consisting of 5 or 6 U-shape, g-s 
drying tubes (or equiv.) joined in series. Fill first tube with 
H 2 S0 4 and second with Ag 2 S0 4 -H 2 S04 soln (10 g Ag 2 S0 4 in 
100 ml H 2 S0 4 ) to remove acidic gases other than C0 2 . Fill 
third tube with Mg(C10 4 ) 2 to absorb H 2 0. Fill inlet 2 / 3 of fourth 
and succeeding tubes with Ascarite to absorb C0 2 , and outlet 
V,3 of each tube with Mg(C10 4 ) 2 . Connect last tube in train with 
aspirating bottle or suction source. 

Condition app. daily before use, and also when freshly filled 
tube is placed in train, by aspirating air at rate of 2-3 bubbles/ 
sec thru dry alkalimeter assembly and absorption train until 
C0 2 absorption tubes attain const wt (usually 20-30 min). Tare 
against similarly packed tubes. Use std procedure for wiping 
tubes with dry, lint-free cloth before each weighing. 

B. Determination 

Transfer 3 g burnt or hydrated lime or 0.5-1.0 g limestone 
or marl, prepd as in 924.03, to dry alkalimeter flask. Mo- 
mentarily open stopcocks of first 2 C0 2 absorption tubes to 
air to equalize pressure, weigh tubes Sep., and place in posi- 
tion in train. With assembled alkalimeter connected to ab- 
sorption train, adjust rate of aspiration of air thru system to ca 
2 bubbles /sec. Close funnel stopcock, remove alkalimeter guard 
tube, fill funnel with 50 mL HC1 (1 +4), and replace guard 
tube. Open funnel stopcock and let acid run slowly into flask, 
taking care that evolution of gas is so gradual as not to ma- 
terially increase flow thru tubes. After all acid is added, agitate 
alkalimeter assembly to ensure complete dispersion of sample 
in acid soln. Continue aspiration, gradually heat contents of 
flask to bp, and boil 2-3 min after H 2 begins to condense. 
Discontinue heating, and continue aspiration 15-20 min or un- 
til app. cools. Remove, equalize internal and external pres- 
sure, and re weigh absorption tubes. 

Increase in wt = wt C0 2 . (Material increase in wt of second 
tube usually indicates exhaustion of first tube, but may result 
from too rapid evolution of C0 2 in relation to aspiration rate.) 
Report % CaC0 3 . 

Ref.: JAOAC 38, 413(1955). 

CAS- 124-38-9 (carbon dioxide) 



CALCIUM SILICATE SLAGS 

944.01 Neutralizing Value for 

Calcium Silicate Slags 
Titrimetric Method 
Final Action 1965 

(Uncorrected for sulfide content) 

(a) Blast furnace slag. — Transfer 0.5 g sample, ground to 
pass No. 80 sieve, to 250 mL erlenmeyer. Wash down with 
small portions H 2 and add 35 mL 0.57V HC1 while swirling. 
Heat to gentle boil over burner, agitating suspension contin- 
uously until bulk of sample dissolves. Boil 5 min and cool to 
room temp.; then dil. with C0 2 -free H 2 to ca 150 mL and 
add 1 mL 30% H 2 2 and 5 drops bromocresol green. (Dissolve 
0.1 g tetrabromo-m-cresolsulfonphthalein in 1.5 mL 0.1 NaOH, 
and dil. to 100 mL with H 2 0.) Back-titr. with 0.5N NaOH, 



adding first 15 mL rapidly and titrg drop wise thereafter, vig- 
orously agitating contents of stoppered flask after each addn, 
until indicator tint matches or slightly exceeds that of pH 5.2 
phthalate buffer soln, 941. 17C, of like vol. and indicator concn, 
after 2-3 sec agitation. 

(b) Rock phosphate reduction furnace slag. — Transfer 0.5 
g sample to 250 mL beaker. Wash down with small portions 
H 2 and add, stirring continuously, 50 mL HO Ac (1 + 4). 
Heat to bp and boil 5 min, stirring frequently. Evap. to dryness 
on steam bath. Add 20 mL of the HO Ac, dil. to 150 mL, and 
heat to bp; add NH 4 OH (1 + 1) to distinct yellow of Me red. 
Digest ca 10 min on hot plate. Filter by gravity thru 9 cm 
paper, catching filtrate in 100 X 50 mm lipped Pyrex crystg 
dish; wash beaker 3 times and paper 5 addnl times with neut. 
0.5N NH 4 OAc. Evap. filtrate on hot plate. Adjust heat so bub- 
bles breaking thru viscous surface film are released gently to 
avoid spattering. (To expedite dehydration, repeat treatments 
with 25 mL hot H 2 and evapn 2 or 3 times.) Continue heating 
residue on hot plate until no HOAc odor remains. Heat addnl 
10 min at full heat of hot plate; then ignite 10 min at 550°. 
Cool, wet residue with 15 mL H 2 0, place watch glass over 
dish, and add 25 mL 0.5N HC1 thru lip of dish. Heat 5 min 
over burner at gentle simmer. Rinse watch glass, filter sus- 
pended matter on 9 cm paper, catching filtrate in 250 mL er- 
lenmeyer, and wash dish and filter 3 times with hot H 2 0. Titr. 
excess acid with 0.5N NaOH to distinct yellow of Me red. 

Net acid used x 5 = neutzg value of slag in terms of % 
CaC0 3 equivalence. 

Refs.: JAOAC 27, 74, 532(1944); 28, 310(1945); 31, 71(1948). 

CAS-471-34-1 (calcium carbonate) 



948.01 Sulfide Sulfur in 

Calcium Silicate Slags 
Titrimetric Method 
Final Action 1965 

(Note: CdS0 4 is toxic; see also safety note on toxic dusts.) 

A. Reagents 

(a) Zinc dust. — Low in Pb. 

(b) Absorbent soln.— Dissolve 20 g CdS0 4 .2 2 /3H 2 in H 2 
and dil. to 1 L. Adjust to pH 5.6 potentiometrically or col- 
orimetrically. If colorimetrically, match sep. 50 mL aliquot to 
buffer of same pH, 941. 17C. 

(c) Sodium hydroxide std soln. — 0.17V. Prep, and stdze as 
in 936.16. 

(d) Std acid.— AN HC1. Stdze against std alkali, (c), us- 
ing Me red. 

(e) Methyl red indicator. — Dissolve 0.2 g Me red in 100 
mL alcohol. 

B. Apparatus 

Fit 250 mL erlenmeyer with 2-hole No. 5.5 stopper. Insert 
thru stopper 60 mL separator with stem drawn out to 2 mm 
and bent upward at tip, adjusting separator so stem is 6 mm 
from bottom of flask. Also insert thru stopper 6 mm glass out- 
let tube. Connect with amber rubber tubing to inlet of 25 x 
150 mm tube half filled with H 2 and heated to near bp before 
and during detn. Connect in series 2 addnl tubes of same size, 
each contg 25 mL absorbent soln and held in 600 mL beaker 
filled with cold H 2 0. 

C. Determination 

Fill absorbent tubes with absorbent soln and heat H 2 tube 
to gentle boiling. Weigh I g slag, ground to pass No. 80 sieve, 



Agricultural Liming Materials 



AOAC Official Methods of Analysis (1990) 



into evolution flask, add 1 g Zn dust, and wash down sides 
with 5-10 mL H 2 0; mix with flat-end rod and connect flask 
to app. Add 50 mL HC1 (1 + 4) to separator and let acid flow 
into reaction flask while swirling contents. If necessary, apply 
pressure to transfer acid and close stopcock while a little of 
the acid is still above it. Heat to bp; then regulate to maintain 
active but not too vigorous boiling for 10 min. Swirl flask 
frequently after adding acid and for first 5 min of boiling. To 
disconnect, hold inlet in first absorbent tube firmly with one 
hand and quickly pull off rubber tubing with other hand with- 
out pinching. 

Filter CdS suspension by gravity thru 9 cm paper into 250 
mL erlenmeyer and wash with H 2 to vol. of 100 mL. Add 
4 drops Me red indicator and agitate vigorously while titrg 
slowly with 0.1N NaOH to exact tint of ref. soln (50 mL ab- 
sorbent soln dild to 100 mL, with identical indicator concn, 
in 250 mL erlenmeyer). If end point is passed so that Cd(OH) 2 
ppts, add 1-2 mL O.lvV HO, let stand until ppt disappears, 
and complete titrn dropwise, agitating vigorously. 

% CaC0 3 equivalence of sulfide S in sample 

- net mL 0.17VNaOH/2 

g Sulfide S/detn - mL 0.LV NaOH X 0.0016 
% Sulfides- g sulfides x 100 

Refs.: J AOAC 31, 715(1948); 32, 73(1949). 

CAS-7704-34-9 (sulfur) 

GRAVIMETRIC ELEMENTAL ANALYSES 

963.01 Elemental Analysis of 

Liming Materials 

Preparation of Sample Solution 

First Action 1963 
Final Action 1965 

(Caution: See safety notes on wet oxidation, nitric acid, 
and perchloric acid.) 

Prep, samples as in 924.03, preferably in agate mortar. Grind 
silicates to pass No. 100 sieve, and dry all samples at 105°. 

Weigh 2 g limestone or 0.5 g silicate. If sample contains 
org. matter, transfer to Pt crucible and place in cold furnace. 
Raise temp, gradually to 1000° and hold 15 min. Transfer sam- 
ple to 400 mL beaker and, if ignited, moisten cautiously with 
H 2 0. Add 10 mL HN0 3 and evap. on hot plate at low heat 
until mixt. becomes pasty. Cool, and add 10 mL H 2 and 20 
mL 60% HC10 4 . Boil to heavy fumes of HCIO4, cover, and 
fume slowly until soln is colorless or slightly yellow (5-10 
min). Do not evap. to dryness. Cool to <100° and add 50 mL 
H 2 0. Filter thru Whatman 41H or finer paper into 250 mL vol. 
flask. Wash thoroly with hot H 2 to remove all traces of HCIO4. 
Reserve filtrate and washings for prepn of Sample Solns X and 
Y, 963.02 

963.02 Silica in Liming Materials 

Gravimetric and Titrimetric Methods 

First Action 1963 
Final Action 1965 

(See also 965.07.) 

(Caution: See safety notes on hydrofluoric acid and 
perchloric acid.) 

Transfer paper with Si0 2 to uncovered Pt crucible and heat 
gently with low flame until paper chars without flame. Par- 



tially cover crucible and cautiously burn C. Finally cover com- 
pletely and heat with blast lamp or in furnace at 1150-1200°. 
Cool in desiccator and weigh. Repeat to const wt (W). Treat 
with ca 1 mL H 2 0, 2 drops H 2 S0 4 (1 + 1), and 10 mL HF. 
Cautiously evap. to dryness in hood. Heat 2 min at 1050— 
1100°, cool in desiccator, and weigh (B). 

W — B ~ g Si0 2 in sample 
g Si0 2 X 0.4674 = g Si 

(a) Sample Soln X.— (0.008 g limestone or 0.002 g silicate/ 
mL.) Fuse residue from Si detn with 0.5 g Na 2 C0 3 by heating 
covered crucible 10 min over Meker burner. Cool, fill crucible 
2 / 3 full with H 2 0, and add 2 mL 60% HC10 4 dropwise, with 
stirring. Warm if necessary to dissolve melt. Add to filtrate 
and washings reserved for prepn of Sample Soln X in 963.01. 
Dil. to 250 mL with H 2 0. 

(b) Sample Soln Y.— (0.00016 g limestone or 0.00004 g sil- 
icate/mL.) Dil. 10 mL Sample Soln X to 500 mL with H 2 0. 

Refs.: JAOAC 46, 603(1963); 47, 1019(1964). 

CAS-763 1-86-9 (silicon dioxide) 



917.01 Aluminum, Iron, Phosphorus, 

and Titanium Oxides in Liming Materials 

Gravimetric Method 

Final Action 1965 

(Alternatively, Al, Fe, Mn, P, and Ti may be detd colori- 

metrically as in 965.01, 965.02, 965.03, 965.04, 965.05, and 

965.06.) 

To 125 mL aliquot Soln X from 963.02(a), add 10 mL HC1 
and few drops Me red indicator; heat to gentle boil and add 
NH4OH (1 + 1) until ppt forms and indicator just changes to 
distinct yellow. Boil <2 min and filter rapidly. Wash ppt 6- 
8 times with hot 2% NH 4 N0 3 soln. Return ppt and filter to 
original beaker, add 10 mL HC1, and macerate filter with po- 
liceman. Dil. with H 2 0, heat to dissolve ppt, dil. to ca 200 
mL, and reppt as above. Wash thoroly with the hot NH4NO3 
soln until CI -free. Combine first and second filtrates and save 
for Ca and Mg detns. 

Place ppt in Pt crucible and dry. Ignite gently to oxidize C, 
heat to bright red ca 10 min, cool in desiccator, and weigh in 
covered crucible as Fe 2 3 + A1 2 3 + P 2 5 + Ti0 2 . 

Refs.: U.S. Geol. Survey Bull. 700, p. 106. Ind. Eng. Chem. 
9, 1114(1917). JAOAC 48, 95(1965). 

CAS- 1344-28-1 (aluminum oxide) 
CAS- 1309-37-1 (ferric oxide) 
CAS- 13 14-56-3 (phosphorus pentoxide) 
CAS- 13463-67-7 (titanium dioxide) 



917.02 Calcium in Liming Materials 

Gravimetric and Titrimetric Methods 
Final Action 1965 

Cone, combined filtrates and washings from 917.01 to ca 
50 mL; make slightly aik. with NH 4 OH (1 + 1); while still hot, 
add satd (NH a ) 2 C 2 O a soln dropwise as long as any ppt forms, 
and then enough excess to convert Mg salts also to oxalate. 
Heat to bp, let stand ^3 hr, decant clear soln thru filter, pour 
15-20 mL hot H 2 on ppt, and again decant clear soln thru 
filter. Dissolve any ppt remaining on filter by washing with 
hot HC1 (1+9) into original beaker, wash 6 times with hot 
H 2 0, and then reppt at bp by adding NH 4 OH and a little satd 



AOAC Official Methods of Analysis (1990) 



Elemental Analysis 



(NH4) 2 C 2 04 soln. Let stand as before, filter thru same filter, 
and wash with hot H 2 G until Cl-free, Reserve filtrates and 
washings from both pptns for detn of Mg, 919. GIB. 

Complete detn by one of following methods and report as 
% CaO: 

(a) Ignite ppt in crucible, either over S-free blast lamp, or 
in elec. furnace at 950°, to const wt, cool in desiccator, and 
weigh as CaO. 

(b) Incinerate filter over low flame, mix ignited ppt with 
finely pulverized and dried mixt. of equal parts of (NH 4 ) 2 S0 4 
and NH 4 Cl, and drive off excess sulfate by carefully heating 
upper portion of crucible. Complete ignition, cool in desic- 
cator, and weigh as CaS0 4 . 

(c) Perforate apex of cone; wash CaC 2 4 ppt into beaker 
used for pptn; then wash filter with hot H 2 S0 4 (1 +4), and titr. 
at 85-90° with 0.17V KMn0 4 . 

Refs.: U.S. Geol. Survey Bull. 700, p. 106. Ind. Eng. Chem. 
9, 1114(1917). 

CAS- 1305-78-8 (calcium oxide) 



919.01 Magnesium in Liming Materials 

Gravimetric Method 
Final Action 1965 

A. Reagent 

Phosphate soln. — Dissolve 100 g (NH 4 ) 2 HP0 4 in hot H 2 0, 
dil. to 1 L, and add 5 mL CHC1 3 . 

B. Determination 

To combined filtrates and washings, 917.02, add 2 mL \M 
citric acid, 100 mL NH 4 OH, and 50 mL alcohol. Then add 25 
mL of the phosphate soln, with const stirring, and let stand 
12-24 hr. Filter, wash twice with NH 4 OH (1 +9), and dissolve 
ppt in HN0 3 (1+4), washing soln into original beaker to vol. 
of 100-150 mL. Add 1/10 vol. NH 4 OH and 2 drops of the 
phosphate soln. Stir vigorously and let stand ^3 hr. Filter thru 
gooch, wash with NH 4 OH (1+9), moisten filter with satd soln 
of NHqNOs made slightly ammoniacal, ignite, and weigh as 
Mg 2 P 2 7 . Report as % MgO, Correct wt Mg 2 P 2 O v for co-pptd 
Mn 2 P 2 7 by detg Mn as in 973.55 A. 

Ref.: Washington, "Chemical Analysis of Rocks," 3rd Ed., 
1919, p. .181. 



CAS- 1309-48-4 (magnesium oxide) 



CHELOMETRIC ELEMENTAL ANALYSES 

962.01 Calcium and Magnesium 

in Liming Materials 
EDTA Titrimetric Methods 

First Action 1962 
Final Action 1965 

(Not applicable to samples with high phosphate content or contg 

<2% Mg) 

(Caution: See safety note on cyanides.) 

A. Reagents 

(a) Buffer soln.— pH 10. Dissolve 67.5 g NH 4 C1 in 200 mL 
H 2 0, add 570 mL NH 4 OH, and dil. to 1 L. 

(b) Potassium hydroxide-potassium cyanide soln. — Dis- 
solve 280 g KOH and 66 g KCN in 1 L H 2 0. 



(c) Potassium cyanide soln. — 2%. Dissolve 2 g KCN in 100 
mL H 2 0. 

(d) Eriochrome black T indicator soln. — Dissolve 0.2 g in- 
dicator (HOC 10 H 6 N:NC 10 H4(OH)(NO 2 )SO3Na) (Eastman Ko- 
dak P6361, or equiv.) in 50 mL MeOH contg 2 g NH 2 OH.HCl. 
Store <1 month. 

(e) Magnesium std solns. — 0.25 and 1.00 ing/mL. Dis- 
solve 0.25 and LOO g Mg turnings in HC1 (1 + 10) and dil. 
each to 1 L with double distd H 2 0. 

(f ) Calcium std soln. — 1 mg/mL. Dissolve 2.4973 g CaC0 3 , 
primary std grade, previously dried 2 hr at 285°, in HC1 (1 + 10). 
Dil. to 1 L with double distd H 2 0. 

(g) Calcein indicator. — Grind together 1 g indicator (2',7'- 
bis[[bis(carboxymethyl)amino]methyl]-fluorescein, sodium 
derivative, sodium salt), 10 g charcoal (Norite A is satisfac- 
tory), and 100 g KC1. (Indicator is described in Anal. Chem. 
28, 882 (1956), and is available from Eastman Kodak.) 

(h) Disodium dihydrogen EDTA std solns. — (1) 0.4%. — 
Dissolve 4 g Na 2 H 2 EDTA in 1 L H 2 0. Stdze against std Ca 
and Mg solns. (2) 0.1%. — Prep, as in (J), using 1 g 
Na 2 H 2 EDTA, and stdze against 0.25 mg/mL Mg std soln. 

B. Standardization 

(a) For calcium. — Pipet 10 mL std Ca soln into 300 mL 
erlenmeyer and add 10 mL H 2 0. Add 10 mL KOH -KCN soln 
and ca 35 mg calcein indicator. Using mag. stirrer and arti- 
ficial light, titr. with 0.4% EDTA std soln to disappearance 
of all green. Titr. >3 aliquots and use av. to calc. titer Ca soln 
= 10/mL EDTA soln. 

(b) For magnesium. — Pipet 10 mL 0.25 and 1.00 mg/mL 
Mg std solns into 300 mL erlenmeyers and add 100 mL H 2 0. 
Add 5 mL pH 10 buffer, 2 mL 2% KCN soln, and 10 drops 
eriochrome black T indicator. Using mag. stirrer and artificial 
light, titr. with 0.1. and 0.4% EDTA std solns, resp., until 
color changes permanently from wine red to pure blue. Titr. 
>3 aliquots and use av. to calc. titer Mg soln = 2.5/mL EDTA 
soln, or 10/mL EDTA soln, resp. 

C. Determination 

Dry sample at 1 10° to const wt and cool to room temp. Grind 
to pass No. 60 or 80 sieve and mix thoroly. Accurately weigh 
ca 0.5 g into 250 mL beaker, add 20 mL HC1 (1 + 1), and evap. 
to dryness on hot plate. Dissolve residue in 5 mL HC1 (1 + 10), 
dil. to ca 100 mL with H 2 0, and digest over low flame 1 hr. 
Cool, transfer to 200 mL vol. flask, dil. to vol., mix, and let 
settle or filter. 

(a) For calcium. — Pipet 10 mL aliquot into 300 mL erlen- 
meyer and titr. as in 962.01B(a), observing end point thru soln 
and away from light. % Ca = (Titer EDTA std soln for Ca) 
X mL EDTA std soln x 2/g sample. 

(b) For magnesium. — (For agricultural limestones contg >4% 
Mg.) For Ca + Mg, pipet 10 mL aliquot into 300 mL erlen- 
meyer and titr. with 0.4% EDTA soln as in 962.01B(b). 

% Mg = (Titer EDTA std soln for Mg) x [(mL EDTA std 
soln in Ca + Mg titrn) - (mL EDTA std soln in Ca titrn)] X 
2/g sample. 

(c) For magnesium. — (For agricultural limestones contg 2- 
4% Mg.) Pipet 10 mL aliquot (0.5-1.0 mg Mg) into 300 mL 
erlenmeyer and add exact vol. of 0.4% EDTA soln required 
for Ca detn. Titr. with 0.1% EDTA soln as in 962.01B(b). 

% Mg = (Titer EDTA std soln for Mg) x mL EDTA std 
0.1% soln x 2/g sample. 

Refs.: JAOAC 45, 1(1962); 46, 611(1963); 48, 95(1965); 50, 
190(1967). 

CAS-7440-70-2 (calcium) 
CAS-7439-95-4 (magnesium) 



Agricultural Liming Materials 



AOAC Official Methods of Analysis (1990) 



COLORIMETRIC ELEMENTAL ANALYSES 

965.01 Elemental Analysis 

of Liming Materials 
Preparation of Sample Solution 

First Action 1965 
Final Action 1975 

(Carry reagent blanks thru detn with stds and samples. Treat 

aliquots of blank soln (corresponding to aliquot sizes of sample 

solns taken for analysis) as in Determination for appropriate 

element and correct values for samples accordingly.) 

Det. A.l, Fe, Mn, P, and Ti in solns prepd by HC10 4 diges- 
tion, 963.01 and 963.02, or NaOH fusion, 965.01. Det. Si 
only in soln prepd by NaOH fusion. 

{Caution: See safety notes on perchloric acid, NaOH, and 
KOH.) 

Prep, samples as in 924.03, preferably in agate mortar. Grind 
samples to pass No. 100 sieve and dry at 105°. 

(a) Sample Soln X. — (0.005 g limestone or 0.002 g silicate/ 
mL.) Place 0.5 g limestone or 0.2 g silicate in 75 ml Ni cru- 
cible. If sample contains org. matter, place uncovered crucible 
in cold furnace, raise temp, gradually to 900°, and hold 15 
min. Remove crucible from furnace and let cool. Mix 0.3 g 
KN0 3 with sample and add 1.5 g NaOH pellets. Cover cru- 
cible with Ni cover and heat 5 min at dull redness over gas 
flame. (Do not fuse in furnace.) Remove from flame and swirl 
melt around sides. Cool, add ca 50 mL H 2 0, and warm to 
disintegrate fused cake. Transfer to 150 mL beaker contg 15 
mL 5N HCIO4 (1(60%)+1). Scrub crucible and lid with po- 
liceman, and wash any residue into beaker. Transfer to 100 
mL vol. flask and dil. to vol. (Sample Soln X). (This soln is 
acidic and is normally clear and free of insol . matter. Occa- 
sionally particles of oxidized Ni from crucible appear. When 
this occurs, let particles settle before taking aliquots.) 

(b) Sample Soln y.— (0.00015 g limestone or 0.00004 g sil- 
icate/mL.) Dil. 15 mL limestone Sample Soln X or 10 mL 
silicate Sample Soln X to 500 mL with H 2 0. 

Ref.: JAOAC 47, 1019(1964). 



965.02 Aluminum in Liming Materials 

Colorimetric Method 

First Action 1965 
Final Action 1975 

A. Reagents 

(a) Aluminum std solns. — (1) Stock soln. — 100 |xg Al/mL. 
To 0.1000 g pure Al metal in 30 mL beaker, add 6 mL HC1 

U + l). 

Cover with watch glass and warm gently until Al completely 
dissolves. Dil. to 1 L with H 2 0. (2) Working soln. — 4 |ULg Al/ 
mL. Dil. 20 mL stock soln to 500 mL. 

(b) Aluminon soln. — Dissolve sep. in H 2 0: 0.5 g NH 4 au- 
rintricarboxylate in 100 mL; 10 g acacia (gum arable) in 200 
mL; and 100 g NH 4 OAc in 400 mL. Filter acacia soln. Add 
56 mL HC1 to NH 4 OAc soln and adjust pH to 4.5 with HC1 
or NH4OH. Combine 3 solns and dil. to 1 L with H 2 0. 

(c) Antifoam soln. — Disperse 0.03 g silicone defoamer (Dow 
Corning Corp. Antifoam A) in 100 mL H 2 0. 

(d) Thiogly colic acid soln.— b\\. 1 mL HSCH 2 COOH to 100 
mL with H 2 0. 



B. Preparation of Standard Curve 

Transfer aliquots of std soln contg 0, 4, 20, 40, 60, and 80 
fxg Al to 100 mL vol. flasks and proceed as in detn. Prep, std 
curve by plotting %T against jxg Al on semilog paper. 

C. Determination 

Use Sample SolnX for limestones contg <0.2% or silicates 
contg <0.8% Al and adjust pH of aliquot to 4.5 with NH 4 0H. 
For materials contg greater conens of Al , use Sample Soln Y 
and omit pH adjustment. 

Transfer aliquot (<20 mL contg <80 fig Al) of Sample Soln 
X or Y to 100 mL vol. flask. Dil. to 20 mL with H 2 0. Add 2 
mL thioglycolic acid soln, 0.5 mL antifoam soln, and 10 mL 
aluminon soln. Place flask in boiling H 2 20 min (250 mL 
beaker contg 125 mL H 2 holds 100 mL vol. flask conve- 
niently). Remove flask from H 2 and let cool ca 30 min. Dil. 
to 100 mL with H 2 0. Use |mg Al soln, 965. 02B, to set 100% 
T at 525 nm. Read %T for sample soln and det. |mg Al from 
std curve. Calc. % Al in sample. 

Ref.: JAOAC 47, 1019(1964). 

CAS-7429-90-5 (aluminum) 

965.03 Iron in Liming Materials 

Cotorimetric Method 

First Action 1965 
Final Action 1975 

A. Reagents 

(a) Iron std solns. — (7) Stock soln. — 100 |xg Fe/mL. Dis- 
solve 0.1000 g pure Fe metal in 5 mL 2iV HC1 and dil. to 1 
L with H 2 0. (2) Working soln. — 5 |xg Fe/mL. Dil. 25 mL 
stock soln to 500 mL. 

(b) 2 ,4 ,6-Tripyridyl-s-triazine (TPTZ) soln. — Dissolve 0.500 
g TPTZ in few drops HC1 and dil. to 1 L with H 2 0. 

(c) Hydroxy lamine hydrochloride soln. — Dissolve 50 g 
NH 2 OH.HCl in H 2 0. Add 10 mL TPTZ soln and 0.5 g 
NaC10 4 .H 2 0, and dil. to 500 mL with H 2 0. Transfer to sep- 
arator, add 25 mL nitrobenzene, and shake several min. Let 
phases sep. and discard lower nitrobenzene phase contg Fe. 
Repeat extn 2 or 3 times. 

(d) Acetate buffer soln. — Dissolve 164 g anhyd. NaOAc in 
H 2 0. Add 115 mL HO Ac, 10 mL NH 2 OH.HCl soln, 0.05 g 
TPTZ, and 1 g NaC10 4 .H 2 0, and dil. to 1 L with H 2 0. Trans- 
fer to separator, add 25 mL nitrobenzene, and shake several 
min. Let phases sep. and discard lower nitrobenzene phase. 
Repeat extn 3 or 4 times. 

B. Preparation of Standard Curve 

Treat aliquots of std soln contg 0, 5, 50, and 100 |mg Fe as 
in detn. Prep, std curve by plotting %T against jxg Fe on semi- 
log paper. 

C. Determination 

Use Sample Soln X (<5 mL) for limestones contg <0.05% 
or silicates contg <0.2% Fe and Sample Soln Y for materials 
contg greater conens of Fe. 

Transfer aliquot (< 1 00 \ig Fe) of Sample Soln X or Y to 1 00 
mL vol. flask. Add 3 mL NH 2 OH.HCl soln and 10 mL TPTZ 
soln. Add NH4OH dropwise until Fe derivative remains violet 
on mixing. Add 10 mL buffer soln and dil. to 100 mL. Use 
\xg Fe soln, 965.03B, to set 100% T at 593 nm. Read %T 
for sample soln and det. |xg Fe from std curve. Calc. % Fe in 
sample. 



Ref.: JAOAC 47, 1019(1964). 

CAS-7439-89-6 (iron) 



AOAC Official Methods of Analysis (1990) 



Elemental Analysis 



965.04 Manganese in Liming Materials 

Colorimetric Method 

First Action 1965 
Final Action 1975 

A. Reagents 

(a) Manganese std soln. — 50 \Lg Mn/mL. Dissolve 0.0500 
g pure Mn metal in 20 mL 0.5 N H 2 S0 4 and dil. to 1 L with 
H 2 0. 

(b) Acid mixture.— Add 800 mL HN0 3 and 200 mL H 3 P0 4 
to H 2 and dil. to 2 L. 

B. Preparation of Standard Curve 

Treat aliquots of std soln contg 0, 50, 100, 300, and 500 
jxg Mn as in detn. Prep, std curve by plotting %T against jxg 
Mn on semilog paper. 

C. Determination 

Transfer aliquot (<500 jig Mn) of Sample Soln X to 150 
mL beaker. Add 25 mL acid mixt. and 0.3 g KI0 4 . Bring to 
bp and keep near boiling temp. 10 min after color develops. 
Let cool, transfer to 50 mL vol. flask, dil. to vol., and mix. 
Use jxg Mn soln, 965. 04B, to set 100% T at 525 nm. Read 
%T for sample soln and det. jxg Mn from std curve. Calc. % 
Mn in sample. 

Ret.: JAOAC 47, 1019(1964). 

CAS-7439-96-5 (manganese) 



965.05 Phosphorus in Liming Materials 

Colorimetric Method 

First Action 1965 
Final Action 1975 

(Do not clean glassware with detergents contg P.) 

A. Reagents 

(a) Phosphorus std solns . — ( 1 ) Stock soln . — 1 00 |JLg P/mL . 
Dissolve 0.4393 g KH 2 P0 4 in H 2 and dil. to 1 L. (2) Working 
soln. — 5 |mg P/mL. Dil. 25 mL stock soln to 500 mL. 

(b) Ammonium molybdate soln. — Dissolve 20 g (NH 4 ) 6 * 
Mo 7 24 .4H 2 in 500 mL H 2 0. Add 285 mL H 2 S0 4 , cool, and 
dil. to 1 L with H 2 0. 

(c) Hydrazine sulfate soln. — Dissolve 2 g N 2 H 4 .H 2 S0 4 in 
H 2 and dil. to 1 L. 

B. Preparation of Standard Curve 

Treat aliquots of std soln contg 0, 5, 50, and 75 (xg P as in 
detn. Prep, std curve by plotting %T against (mg P on semilog 
paper. 

C. Determination 

Transfer aliquot (<15 mL contg <75 jxg P) of Sample Soln 
X to 100 mL vol. flask. Add 5 mL NH 4 molybdate soln and 
mix. Add 5 mL N 2 H 4 .H 2 S0 4 soln, dil. to 70 mL with H 2 0, 
and mix. Place flask in boiling H 2 9 min. Remove, cool rap- 
idly, and dil. to vol. Use \Lg P~soln, 965.05B, to set 100% 
T at 827 nm. Read %T for sample soln and det. \xg P from 
std curve. Calc. % P in sample. 

Ref.: JAOAC 47, 1019(1964). 

CAS-7723-14-0 (phosphorus) 



965.06 Titanium in Liming Materials 

Colorimetric Method 

First Action 1965 
Final Action 1975 

A. Reagents 

(a) Titanium std solns. — (/) Stock soln. — 1 00 |xg Ti/mL. 
Place 0.1668 g Ti0 2 and 2 g K 2 S 2 7 in Pt crucible. Heat cov- 
ered crucible gently at first and then at dull red ca 15 min. 
Dissolve melt in 50 mL H 2 S0 4 (1 + 1) and dil. to 1 L with 
H 2 0. (2) Working soln.— 5 \xg Ti/mL. Dil. 25 mL stock soln 
to 500 mL. 

(b) Acetate buffer soln. — pH 4.7. Dissolve 41 g anhyd. 
NaOAc in LLO, add 30 mL HOAc, and dil. to 1 L. 

(c) Disodium-1 ,2-dihydroxybenzene-3 ,5 -disulfonate {Tiron) 
soln. — Dissolve 4 g Tiron in H 2 and dil. to 100 mL. 

B. Preparation of Standard Curve 

Treat aliquots of std soln contg 0, 5, 50, and 75 |xg Ti as 
in detn, but do not add dithionite to stds. Prep, std curve by 
plotting %T against ^g Ti on semilog paper. 

C. Determination 

Transfer aliquot (<75 u.g Ti) of Sample Soln X to 50 mL 
beaker. Dil. to c a 25 mL with H 2 0. Add 5 mL Tiron soln and 
then NH 4 OH (1 +9) dropwise until soln is neut. to Congo Red 
paper. (Tiron soln must be added before pH is adjusted.) 
Transfer to 50 mL vol. flask, add 5 mL buffer soln, dil. to 
vol. with H 2 0, and mix thoroly. Add 25 mg dithionite (Na 2 - 
S 2 4 ) and dissolve with min. agitation (to avoid reappearance 
of blue). Use |xg Ti soln, 965.06B, to set 100% T at 410 
nm. Read %T for sample soln within 15 min after adding di- 
thionite. Det. jxg Ti from std curve. Calc. % Ti in sample. 

Ref.: JAOAC 47, 1019(1964). 

CAS-7440-32-6 (titanium) 



965.07 Silicon in Liming Materials 

Colorimetric Method 

First Action 1965 
Final Action 1975 

(Clean all glassware with HCl (1 + 1).) 

A. Reagents 

(a) Silicon std soln.— 20 |xg Si/mL. Place 0.0428 g pure 
Si0 2 in 75 mL Ni crucible and treat as in 965.01(a), but dil. 
with H 2 to 1 L instead of 100 mL. 

(b) Tartaric acid soln. — Dissolve 50 g tartaric acid in H 2 
and dil. to 500 mL. Store in plastic bottle. 

(c) Ammonium molybdate soln. — Dissolve 7.5 g (NH 4 ) 6 - 
Mo 7 24 .4H 2 in 75 mL H 2 0, add 10 mL H 2 S0 4 (1 + 1), and 
dil. to 100 mL with H 2 0. Store in plastic bottle. 

(d) Reducing soln. — Dissolve 0.7 g Na 2 S0 3 in 10 mL H 2 0. 
Add 0.15 g l-amino-2-naphthol-4-sulfonic acid and stir until 
dissolved. Dissolve 9 g NaHS0 3 in 90 mL H 2 0, add to first 
soln, and mix. Store in plastic bottle. 

B. Preparation of Standard Curve 

Treat aliquots of std soln contg 0, 20, 100, and 200 |mg Si 
as in detn. Prep, std curve by plotting %T against \xg Si on 
semilog paper. 



8 Agricultural Liming Materials AOAC Official Methods of Analysis (1990) 

C. Determination and let stand ^30 min. Use \xg Si soln, 965.07B S to set 

Transfer 10 mL Sample Soln Y to 100 mL voh flask (use 100% T at 650 nm. Read %T for sample soln and det. \xg Si 

Sample Soln X for limestones contg <0.2% Si) and add 1 mL from std curve. Calc. % Si in sample. 

NH 4 molybdate soln with swirling. Mix well, and let stand 10 d ^ . iaOAC 47 1019(1964^ 
min. Add 4 mL tartaric acid soln with swirling, and mix well. 

Add 1 mL reducing soln with swirling, dil. to vol., mix well, CAS-7440-21-3 (silicon) 



2, Fertilizers 

Frank J. Johnson, Associate Chapter Editor 

National Fertilizer Development Center, Tennessee Valley Authority 



929.01 Sampling of Solid Fertilizers 

Finai Action 1974 

(a) Bagged fertilizers. — Use slotted single or double tube 
trier with solid cone tip, constructed of stainless steel or brass. 
(Do not use unplated brass for samples on which micronu- 
trients are to be detd.) Trier length, exclusive of handle, should 
be approx. length of filled bag to be sampled, but >25"; length 
of slot >23"; width of slot ^0.5"; and id >: 5 / 8 ". 

Take sample as follows: Lay bag horizontally and remove 
core diagonally from end to end. From lots of >10 bags, take 
core from each of 10 bags. When necessary to sample lots of 
<10 bags, take 10 cores but at least 1 core from each bag 
present. For small packages (<10 lb), take 1 entire package 
as sample. 

(b) Bulk fertilizers, including railroad car-size lots.- — Use 
trier of design represented in Table 929.01. 

Draw 10 vertical cores distributed in std concentric sampling 
pattern (Fig. 929.01A) of such design that each core represents 
approx. equal fractions of lot. 

Bulk shipments may be sampled at time of loading or un- 
loading by passing sampling cup, Fig. 929. 0IB (mouth di- 
mensions: width 3 / 4 ", length 16" or as long as max. diam. of 
stream), thru entire stream of material as it drops from belt or 
chute. Make sampling such as to assure >10 equal-timed-spaced 
passes thruout transfer operation. Stream samples are not ap- 
plicable unless uniform continuous flow of fertilizer is main- 
tained for >3 min while lot is being sampled. 

(c) Preparation of sample .—Place composite sample in air- 
tight container and deliver entire sample to laboratory. Reduce 
composite sample in laboratory, using riffle. 

Refs.: JAOAC 12, 97(1929); 33, 424(1950); 38, 108,541 
(1955); 50, 190,382(1967); 51, 859(1968); 55, 709 
(1972). 



969.01 Sampling of Liquid Fertilizers 

Final Action 

(In absence of free ammonia) 

(a) Clear so Ins.- — (Mixed liqs and N solns.) Secure sample 
directly from mixing vat, storage tank, or delivery tank after 
thoro mixing. Take sample from surface or thru direct tap. 
Flush direct tap, or delivery line and faucet, and collect sample 
in glass or polyethylene container. Alternatively, lower sample 
container into well mixed material thru port in top of tank and 
let fill. Seal container tightly. 

(b) Fluid fertilizers with suspended material. — (Salt sus- 
pensions and slurries.) Agitate material in storage until thoroly 
mixed (15 min usually adequate) before taking sample. Sam- 
ple directly as in (a), or use 500 mL Missouri or Indiana sam- 
pling bottle, Fig. 969.01. Lower sampling bottle from top 
opening to bottom of tank and raise slowly while filling. Transfer 
to sample bottle and seal tightly. 

Alternatively, secure sample from tap on recirculation line 
after agitating and recirculating simultaneously until thoroly 
mixed. Draw sample while recirculating. If recirculation line 



is attached to manifold delivery line, allowing cross-contam- 
ination, pump ca 30 cm (1') or 2000 L (500 gal.) into tem- 
porary storage tank, then sample from recirculation line as above 
or from delivery line. Transfer to sample bottle and seal tightly. 

Ref.: JAOAC 52, 592(1969). 

959.01 Sampling of Ammoniacal Solutions 

First Action 1959 
Final Action 1960 

A. Apparatus 

(a) Container. — Polyethylene reagent-form bottle with but- 
tress-type cap, 1 L (1 qt) capacity. 

(b) Sample flow control apparatus .—Construct from fol- 
lowing fittings: IV2 x X W reducing bushing; l / 4 " tee; V/ nipple 
12-18" long (length not critical); two 7/ stainless steel, blunt- 
nose needle valves with hose connections (Hoke No. 3712M4Y; 
Hoke Inc., 1 Tenakill Pk, Cresskill, NJ 07626). All fittings 
except valves can be either Al or stainless steel. (See Fig. 
959.01.) 

Attach valves directly to tee, which is then attached to re- 
ducing bushing thru nipple. To both valves attach 1 / 4 " id Tygon 
tubing (Hoke No. 62065 hose connection), 12" length to sam- 
ple valve and sufficient length to vent valve to reach disposal 
area or container. To free end of sample tubing attach 3" length 
of ] /V' glass or stainless steel tubing inserted thru No. 4 rubber 
stopper. To exit end of metal tube attach addnl 6" length of 
Tygon tubing. Make certain all connections are tight. App. can 
be attached directly to tank cars, but requires addnl coupling, 
which varies with installation, to attach to storage tanks. IV2" 
"quick coupler" (Ever-Tite Coupling Co., 254 W 54th St, New 
York, NY 10019) suffices in most cases. 

Ref.: JAOAC 42, 500 (1959). 

B. Sampling 

Prep, sample bottle in laboratory by adding ca 500 mL H 2 0, 
replacing cap, and weighing accurately (±0.1 g). Attach sam- 
pling app. to car or tank and, with sample valve closed, flush 
line thru vent valve. Partially collapse sample bottle, insert 
sample tube with stopper, and seal tightly. With sample tube 
dipping below surface of H 2 in bottle, throttle vent valve to 
maintain small flow of soln and partially open sample valve, 
collecting ca 100 mL sample. (Bottle should not expand to full 
size during this time.) Close sample valve, remove sample tube, 
partially collapse bottle, and cap tightly. Reweigh (±0.1 g) 
and calc. wt sample. Cool to 20°, transfer to 1 or 2 L vol. 
flask, dil. to vol. with H 2 0, mix thoroly, and take aliquots for 
analysis. 

959.02 Sampling of Anhydrous Ammonia 

First Action 1959 
Final Action 1960 

(Caution: Use extreme care in handling anhyd. NH 3 . Suitable 

gas mask and rubber gloves are required. See safety note on 

ammonia. ) 



10 



Fertilizers 



AOAC Official Methods of Analysis (1990) 



Table 929.01 


Trier Specifications 








Trier 


Length, in. 


od, in. 


id, in. 


Compartments 
No. Size, in. 


Missouri 
552 Grain* 
Missouri "D" b 


59 
63 
49 


17« 

1 : Vs 

1% 


7s 
17s 
1 


8 

11 

1 


3 

37 a 
43 



Triers available from: 

a Seedburo Equipment Co., 1022 W Jackson Blvd, Chicago, IL 60607. 

"Boyt Tool & Die Co., 917 Maple St, West Des Moines, IA 50265. 



A. Sampling 

Use sample tube of thermal shock-resistant glass calibrated 
to contain 100 mL and graduated in 0.05 mL subdivisions up 
to 0.5 mL. (Dupont special oil centrf. tube or ASTM long- 
form oil tube is satisfactory.) Flush line and fill tube to 100 
mL mark with sample in such manner that condensing mois- 
ture will not enter sample tube. (Skirt attached to end of sam- 
ple line will drain moisture away.) 

B. Water and Nitrogen 

Immediately close sample tube with tight-fitting rubber stop- 
per into which is inserted tight-fitting piece of 6 mm id glass 
tubing 5™ 8 cm long, bent at its exit from outer end of stopper 
to let gases escape but to exclude entrance of moisture or mois- 
ture-laden air. Place in H 2 bath at approx. air temp, and let 
NH 3 evap. When temp, of sample tube is ca that of bath, re- 
move tube, wipe outer surface, and det. vol. of residue. 

% H 2 in sample = mL residue x C 

where C = 0.74, 0.70, or 0.66 for pressures in original con- 
tainers of 100, 150, or 200 psi, resp. 

% N - (100 - % H 2 0) x 0.8224 

Ref.: JAOAC 42, 500(1959). 

CAS-7727-37-9 (nitrogen) 



7 


<^ 4 ^*** 


8 


[3 


© 


5 ) 


10 


^. 6 ^ 


9 



FIG. 929.01 A— Sampling pattern 



OPEN 
MOUTH 




s Air escape hole 1/8" 

-Fluid intake tube: 1/4" for solutions 

3/8" for suspensions 
and slurries 
Nylon cord 



500 mL polyethylene 
bottle 




700 g of lead wts 
(may be placed 
inside or 
attached 
outside of 
bottle) 



MISSOURI 



Air escape hole 3 mm 
Fluid intake tube 7 mm 
Stainless steel jacket 




INDIANA 



FIG. 969.01 — Missouri and Indiana weighted restricted-fill fluid 

fertilizer sampling bottles designed to fill while being lowered 

(and raised) in storage tanks 

929.02 Preparation of Fertilizer Sample 
Final Action 

Reduce gross sample to amt sufficient for analysis or grind 
>225 g (0.5 lb) of reduced sample without previous sieving. 



NEEDLE VALVE - BLUNT 
NEEDLE 




FIG. 929.01 B— Sampling cup 



COUPLER 

FIG. 959.01— Sampling apparatus for ammoniacal solutions, 
including "quick coupler" for attaching to storage tanks 



AOAC Official Methods of Analysis (1990) 



Water 



11 



For fertilizer materials and moist fertilizer mixts, grind to pass 
sieve with 1 mm circular openings, or No. 20 sieve; for dry 
mixts that tend to segregate, grind to pass No. 40 sieve. Grind 
as rapidly as possible to avoid loss or gain of moisture during 
operation. Mix thoroly and store in tightly stoppered bottles. 

Refs.: JAOAC 12, 98(1929); 24, 253(1941). 



917.03* Bone, Tankage, 

and Basic Slag Fertilizers 
Mechanical Analysis 

Final Action 
Surplus 1970 

See 2.008, 11th ed. 



957.01 Phosphate Rock Fertilizers 

Mechanical Analysis 
Final Action 

A. Apparatus 

(a) Water pressure control. — See Fig. 957.01. Connect 
valve, A, std pressure gage, B, and aerator, C, with 3 /s" diam. 
pipe. 

(b) Sieves. — Nos. 100 and 200, bronze or stainless steel 
cloth, checked against certified sieves. Sieves 8" diam. and 2" 
in depth to sieve cloth are recommended for both wet and dry 
sieving, but other sizes may be used if detd to be suitable un- 
der conditions of method. (Other sieves in U.S. series may be 
used, with precaution to ensure complete sepn of sample into 
desired fractions.) 

(c) Sieve shaker. — Ro-Tap (C-E Tyler, Inc., 3200 Besse- 
mer City Rd, Hwy 274, PO Box 8900, Gastonia, NC 28053), 
Syntron (FMC Corp., Material Handling Equipment Div., Ho- 
mer City, PA 15748), or other suitable machine. 

B. Reagent 

Dispersing agent. — Dissolve 36 g Na hexametaphosphate 
and 8 g Na 2 C0 3 in H 2 and dil. to I L. 



C. Determination 

(a) Ground phosphate rock. — Place 100 g sample on No. 
200 sieve and wash with moderate stream of tap H 2 at max. 
gage pressure of 0.28 kg/sq cm (4 Jb/sq in.) until H 2 passing 
sieve is clear, with care to avoid loss of sample by splashing. 
Dry material remaining on sieve at 105° and transfer to No. 
100 sieve in series with No. 200 sieve of same diam. and depth. 
Shake 8 min in mech. shaker. Det. % sample passing No. 100 
sieve by subtracting wt of material retained on that sieve from 
100. Det. % sample passing No. 200 sieve by subtracting sum 
of wts of material retained on that sieve and on No. 100 sieve 
from 100. 

(b) Soft phosphate with colloidal clay. — Add 100 g sample 
to rapidly stirred soln of 50 mL dispersing agent and 450 mL 
tap H 2 0, with care to avoid contact of un wetted material with 
shaft of stirrer and side of beaker. Stir 5 min after addn of 
sample is completed. Transfer slurry to No. 200 sieve and pro- 
ceed as in (a). 

Ref.: JAOAC 40, 711(1957). 



955.03* Ash (Acid-Insoluble) of Fertilizers 

Final Action 
Surplus 1970 



See 2.015, 1 1th ed. 



WATER 

950.01 Water (Total) in Fertilizers 

Final Action 



(Not applicable to samples that yield volatile substances other 
than H 2 at drying temp.) 

Heat 2 g sample, 929.02, 5 hr in oven at 100±l o . In case 
of NaN0 3 , (NH 4 ) 2 S0 4 , and K salts, heat to const wt at I30±l°. 
Report % loss in wt as H 2 at temp. used. 

Ref.: JAOAC 33, 260(1950). 




O^ 



3/8" waterpipe 



FIG. 957.01 — Apparatus for control of water pressure 



965.08 Water (Free) in Fertilizers 

Vacuum-Desiccation Methods 

First Action 1965 
Final Action 1974 

(Caution: See safety note on magnesium perchlorate.) 

A. Method I 

Place 2 g prepd sample, 929.02, in tared weighing dish. 
(Weigh extremely hygroscopic or damp materials by differ- 
ence in covered dishes.) Dry sample at 25-30° (precise results 
depend on as const a temp, as possible) in vac. desiccator over 
anhyd. Mg(C10 4 ) 2 , P 2 5 , or BaO, under ^50 cm (20") or <55 
cm (22") vac. (20-25 cm (8-10") absolute pressure) 16-18 
hr. Reweigh, and report % loss in wt as free H 2 0. 

B. Method // 

(Not applicable to samples which yield volatile substances other 
than H 2 0) 

Weigh 2 g prepd sample, 929.02, into tared glass weighing 
dish. Dry sample 2 hr± 10 min at 50± 1 .5° in oven under vac. 
of 48-53 cm (19-21") (23-28 cm (9-1 1") absolute pressure). 
(Temp, control within specified limits thruout oven chamber 



12 



Fertilizers 



AOAC Official Methods of Analysis (1990) 



is essential.) Maintain vac. by passing desiccated air thru 
chamber. Coo] dried sample in desiccator and re weigh. Report 
% loss in wt as free H 2 0. 

Refs.: JAOAC 46, 582(1963); 47, 32, 1040(1964); 48, 98 
(1965). 



972.01 Water (Free) in Fertilizers 

Alternative Extraction Method 

First Action 1972 
Final Action 1974 

A. Principle 

Free H 2 is extd with dioxane and detd by titrn with Karl 
Fischer reagent. 

B. Reagents 

(Keep exposure of org. reagents to air at min.) 

(a) Karl Fischer reagent. — Stabilized single soln (Fisher 
Scientific Co., So-K-3, or equiv.) dild ca 1 + 1 with stabi- 
lized diluent (Fisher, So-K-5, or equiv.), or soln equiv. to 2.5 
mg H 2 0/mL. Stdze daily with ca 0.2 g Na tartrate. 2H 2 0. 1 
mg Na tartrate. 2H 2 = 0.1566 mg H 2 0. 

(b) Methanol. — Low in H 2 0. 

C. Determination 

Accurately weigh 2.5 g prepd sample, 929.02, into 125 mL 
erlenmeyer, add 50.0 mL 1 ,4 -dioxane, stopper, mix by swirl- 
ing, and let stand 15 min. Mix thoroly by swirling, and centrf. 
in closed tube. {Caution: See safety note on centrifuges.) 

Transfer 10 mL aliquot to titrn vessel contg pretitrd MeOH 
and titr. with Karl Fischer reagent. (Discard contents of titrn 
vessel after 3 titrns, replace with enough MeOH to cover elec- 
trodes, and pretitr. before proceeding with next sample.) Det. 
blank on 10 mL dioxane as above and subtract from sample 
detns. Calc. and report as free H 2 0. 

Refs.: JAOAC 52, 1127(1969); 55, 699(1972). 



PHOSPHORUS 

957.02 Phosphorus (Total) in Fertilizers 
Preparation of Sample Solution 
Final Action 

A. Reagent 

Magnesium nitrate soln. — Dissolve 950 g P-free 
Mg(N0 3 ) 2 .6H 2 in H 2 and dil. to 1 L. 

B. Preparation of Solution 

{Caution: See safety notes on wet oxidation, nitric acid, per- 
chloric acid, sulfuric acid, and oxidizers.) 

Treat 1 g sample by (a), (b), (c), (d), or (e), as indicated. 
Cool soln, transfer to 200 or 250 mL vol. flask, dil. to vol., 
mix, and filter thru dry filter. 

(a) Materials containing small quantities of organic mat- 
ter. —Dissolve in 30 mL HN0 3 and 3-5 mL HC1, and boil 
until org. matter is destroyed (30 min for liqs and suspen- 
sions). 

*(b) Fertilizers containing much Fe or Al phosphate, and 
basic slag.*— See 2.017, 10th ed. 

(c) Organic material like cottonseed meal alone or in mix- 
tures. — Evap. with 5 mL Mg(N0 3 ) 2 soln, 957. 02 A, ignite, 
and dissolve in HC1. 



*(d) Materials or mixtures containing large amounts of or- 
ganic matter.* — See 2.017(d), 11th ed. 

(e) All fertilizers . — Boil gently 30-45 min with 20-30 mL 
HN0 3 in suitable flask (preferably Kjeldahl for samples contg 
large amts of org. matter) to oxidize all easily oxidizable mat- 
ter. Cool. Add 10-20 mL 70-72% HC10 4 . Boil very gently 
until soln is colorless or nearly so and dense white fumes ap- 
pear in flask. Do not boil to dryness at any time (Danger!). 
(With samples contg large amts of org. matter, raise temp, to 
fuming point, ca 170°, over period of ^1 hr.) Cool slightly, 
add 50 mL H 2 0, and boil few min. 

Ref.: JAOAC 40, 690(1957). 

CAS-7723-14-0 (phosphorus) 



958.01 Phosphorus (Total) in Fertilizers 

Spectrophotometry Molybdovanadophosphate Method 
Final Action 

(Not applicable to materials yielding colored solns or solns 
contg ions other than orthophosphate which form colored com- 
plexes with molybdo vanadate. Not recommended for basic 

slag.) 

A. Apparatus 

Photometer. — Spectrophtr with stray light filter and matched 

1 cm cells. Analyst must det. suitability for use and conditions 
for satisfactory performance. Means for dispelling heat from 
light source is desirable. 

B. Reagents 

(a) Molybdovanadate reagent. — Dissolve 40 g NH 4 molyb- 
date.4H 2 in 400 mL hot H 2 and cool. Dissolve 2 g NH 4 
metavanadate in 250 mL hot H 2 0, cool, and add 450 mL 70% 
HCIO4. {Caution: See safety notes on perchloric acid.) Grad- 
ually add molybdate soln to vanadate soln with stirring, and 
dil. to 2 L. 

(b) Phosphate std soln.— Dry pure KH 2 P0 4 (52. 15% P 2 5 ) 

2 hr at 105°. Prep, solns contg 0.4-1.0 mg P 2 5 /mL in 0.1 
mg increments by weighing 0.0767, 0.0959, 0.1151, 0.1342, 
0.1534, 0.1726, and 0.1918 g KH 2 P0 4 and dilg each to 100 
mL with H 2 0. Prep, fresh solns contg 0.4 and 0.7 mg P 2 5 / 
mL weekly. 

C. Preparation of Standard Curve 

Pipet 5 mL aliquots of 7 std phosphate solns (2-5 mg P 2 5 / 
aliquot) into 100 mL vol. flasks and add 45 mL H 2 0. Then, 
within 5 min for entire series, add 20 mL molybdovanadate 
reagent by buret or pipet, dil. to vol. and mix. Let stand 10 
min. 

Select 2 absorption cells (std and sample cells) and fill both 
with 2 mg std. Set spectrophtr to 400 nm and adjust to zero 
A with std cell. Sample cell must check zero A within 0.001 
unit; otherwise read A for sample cell and correct subsequent 
readings. (Choose cell showing pos. A against other as sample 
cell so that this pos, A is always subtracted.) Using sample 
cell, det. A of other stds with instrument adjusted to zero A 
for 2 mg std. After each detn empty and refill cell contg 2 mg 
std, and readjust zero to avoid error that might arise from temp. 
changes. Plot A against concn in mg P 2 5 /mL std soln. 

D. Preparation of Solution 

Treat 1 g sample as in 957. 02B, preferably (e), when these 
acids are suitable solv. (Soln should be free of N oxides and 
NOC1.) 

(a) For P 2 5 content <5%, dil. to 250 mL. 



AOAC Official Methods of Analysis (1990) 



Phosphorus 



13 



(b) For P 2 O s content >5%, dil. to such vol. that 5 or 10 
mL aliquot contains 2-5 mg P 2 5 . 

E. Determination 

Pipet, into 100 mL vol. flasks, 5 mL aliquots of std phos- 
phate solns contg 2 and 3.5 mg P 2 5 /aliquot, resp., and de- 
velop color as in 958. 01C. Adjust instrument to zero A for 2 
mg std, and det. A of 3.5 mg std. (It is essential that A of 
latter std be practically identical with corresponding value on 
std curve.) 

(a) Samples containing up to 5% P 2 5 . — Pipet, into 100 
mL vol. flask, 5 mL sample soln, 958.01D(a), and 5 mL std 
phosphate soln contg 2 mg P 2 5 . Develop color and det. A 
concurrently with and in same manner as for std phosphate 
solns in preceding par., with instrument adjusted to zero A for 
2 mg std. Read P 2 5 concn from std curve. With series of 
sample solns, empty and refill cell contg 2 mg std after each 
detn. 



% P 2 5 in sample - 100 

X [(mg P 2 O s from std curve - 2)/20] 

(b) Samples containing more than 5% P 2 5 . — Pipet 5 or 
10 mL sample soln, 958.01D(b), into 100 mL vol. flask. 
Without adding std phosphate soln, proceed as in (a). 

% P 2 O s in sample 

= 100 x (mg P 2 5 from std curve/mg sample in aliquot) 

Refs,: JAOAC 41, 517(1958); 42, 503(1959); 44, 133(1961). 
CAS- 13 14-56-3 (phosphorus pentoxide) 



962.02 Phosphorus (Total) in Fertilizers 

Gravimetric Quinolinium Molybdophosphate Method 

First Action 1962 
Final Action 1965 

A. Reagents 

(Store solns in polyethylene bottles.) 

(a) Citric-molybdic acid reagent. — Dissolve 54 g 100% 
molybdic anhydride (Mo0 3 ) and 12 g NaOH with stirring in 
400 mL hot H 2 0, and cool. Dissolve 60 g citric acid in mixt. 
of 140 mL HC1 and 200 mL FLO, and cool. Gradually add 
molybdic soln to citric acid soln with stirring. Cool, filter, and 
dil. to 1 L. (Soln may be green or blue; color deepens on 
exposure to light.) If necessary, add 0.5% KBr0 3 soln drop- 
wise until green color pales. Store in dark. 

(b) Quinoline soln. — Dissolve 50 mL synthetic quinoline, 
with stirring, in mixt. of 60 mL HC1 and 300 mL H 2 0. Cool, 
dil. to 1 L, and filter. 

(c) Quimociac reagent. — Dissolve 70 g Na molybdate. 
2H 2 in 150 mL H 2 0. Dissolve 60 g citric acid in mixt. of 
85 mL HN0 3 and 150 mL H 2 0, and cool. Gradually add mo- 
lybdate soln to citric acid-HN0 3 mixt. with stirring. Dissolve 
5 mL synthetic quinoline in mixt. of 35 mL HN0 3 and 100 
mL H 2 6. Gradually add this soln to molybdate-citric acid-HN0 3 
soln, mix, and let stand 24 hr. Filter, add 280 mL acetone, 
dil. to 1 L with H 2 0, and mix. 

B. Preparation of Solution 

Treat 1 g sample as in 957. 02B, dilg to 200 mL. 

C. Determination 

Pipet, into 500 mL erlenmeyer, aliquot contg <25 mg P 2 O s 
and dil. to ca 100 mL with H 2 0. Continue by one of the fol- 
lowing methods: 



(a) Add 30 mL citric-molybdic acid reagent and boil gently 
3 min. (Soln must be ppt-free at this time.) Remove from heat 
and swirl carefully. Immediately add 10 mL quinoline soln 
from buret with continuous swirling. (Add first 3-4 mL drop- 
wise and remainder in steady stream.) Or: 

(b) Add 50 mL quimociac reagent, cover with watch glass, 
place on hot plate in well- ventilated hood, and boil 1 min. 

After treatment by (a) or (b), cool to room temp., swirl 
carefully 3-4 times during cooling, filter into gooch with glass 
fiber filter paper previously dried at 250° and weighed, and 
wash with five 25 mL portions of H 2 0. Dry crucible and con- 
tents 30 min at 250°, cool in desiccator to room temp., and 
weigh as (C9H 7 N) 3 H3[P04.12Mo0 3 ]. Subtract wt reagent blank. 
Multiply by 0.03207 to obtain wt P 2 5 (or by 0.01400 for P). 
Report as % P 2 5 (or % P). 

Refs.: Z. Anal. Chem. 189, 243(1962). JAOAC 45, 40, 999 
(1962); 46, 579(1963); 47, 420(1964). 

CAS-7723-14-0 (phosphorus) 

CAS- 13 14-56-3 (phosphorus pentoxide) 



969.02 Phosphorus (Total) in Fertilizers 

Alkalimetric Quinolinium Molybdophosphate Method 

First Action 1969 
Final Action 1975 

A. Reagents 

(a) Quimociac reagent. — See 962.02A(c). 

(b) Sodium hydroxide std soln. — (1 mL — 1 mg P 2 5 .) Dil. 
366.32 mL IN NaOH, 936.16, to 1 L with H 2 0. 

(c) Nitric acid std soln. — Prep. HNO3 soln equiv. to concn 
of (b) and stdze by titrg against (b), using phthln. (For greater 
precision, use HNO3 soln corresponding to 1/5 concn of (b).) 

(d) Citric acid. — \0% (w/v). 

(e) Indicators. — (J) Thymol blue soln. — 0.1%. Add 2.2 mL 
0AN NaOH to 0. 1 g thymol blue and dil . to 100 mL with 50% 
alcohol. (2) Phenolphthalein. — 0.1%. Dissolve 0.1 g phthln 
in 100 mL 50% alcohol. (3) Mixed indicator. — Mix 3 vols (/) 
and 2 vols (2). 

B. Preparation of Sample Solution 

Treat 1 g sample as in 957.02B. 

C. Determination 

(a) Precipitation. — Transfer aliquot contg <30 mg P 2 5 and 
^5 mL coned acid to 500 mL erlenmeyer, add 20 mL citric 
acid soln, and adjust to ca 100 mL. Add 60 mL quimociac 
reagent, immediately cover with watch glass, and place on me- 
dium temp, hot plate. After soln comes to bp, move to cooler 
portion of hot plate and boil gently 1 min. Let cool until flask 
can be handled comfortably with bare hand. 

(b) Filtration and washing. — Prep, pulped-paper pad ca 6 
mm thick on perforated porcelain disk in funnel by adding >:2 
approx. equal increments of H 2 suspension of pulped paper 
and sucking dry with vac. between addns. Swirl flask, pour 
contents onto filter, and wash flask with five ca 15 mL por- 
tions H 2 0, adding washings to funnel. Immediately after fun- 
nel has emptied, wash down sides with ca 15 mL H 2 to re- 
move residual acetone, which causes excessively fast drying 
and later lump formation if allowed to evap. Wash with 3 addnl 
15 mL portions H 2 0, letting funnel empty between addns. Keep 
drying of ppt to min. Using only jet of H 2 0, transfer ppt and 
pad to pptn flask and break up pad with jet of H 2 0. Do not 
smear ppt against funnel or flask. 

(c) Titration. — Titr. with std NaOH soln and add 3-5 mL 
excess. Add 1 mL mixed indicator and titr. with std HNO3 



14 



Fertilizers 



AOAC Official Methods of Analysis (1990) 



soln to grey-blue end point. If overtitrd (greenish-yellow), add 
addnl excess std NaOH soln and titr. to grey-blue. 

(d) Blank. — Det. blank on all reagents, adding known amt 
(1-2 mg) of P 2 5 . Use 1 + 9 dilns of std NaOH and HN0 3 
for titrn and subtract theoretical titer equiv. to P 2 5 added from 
experimental titer. Calc. difference equiv. to 0.36637V NaOH 
and subtract this blank from all sample detns. 

Calc. and report as % P 2 5 . 

Refs.: Z. Anal. Chem. 189, 243(1962). JAOAC 45, 40, 999 
(1962); 49, 1201(1966); 52, 587(1969). 

CAS- 13 14-56-3 (phosphorus pentoxide) 

978.01 Phosphorus (Total) in Fertilizers 

Automated Method 

First Action 1978 
Final Action 1980 

A. Principle 

Samples are extd for direct available P 2 5 or for total P2O5 
detns. Destruction of coloring matter, hydrolysis of nonortho- 
phosphates, and elimination of citrate effect are accomplished 
by digestion with AN HC10 4 at 95°. Digested samples are re- 
acted with molybdovanadate reagent, and A of resulting com- 
plex is read in flowcell at 420 nm in range 0.15-0.35 mg 
P 2 O s /mL. 

B. Apparatus and Reagents 

(Caution: See safety notes on perchloric acid.) 

(a) Automatic analyzer.— Auto Analyzer with following 
modules (Technicon Instruments Corp., or equiv.): Sampler II 
or IV with 40/hr (4:1) cam; proportioning pump III; P 2 O s anal. 



cartridge (with 2 heating baths, each contg 10.6 mL coil held 
at 95±1°; or AAI type heating bath contg one 40' x 1.6 mm 
id coil and holding constant temp, of 95±.l.°); A All single 
channel colorimeter with 15 x 1.5 or 2.0 mm id flowcell and 
420 nm interference filters; voltage stabilizer; and recorder. 
Construct manifold as in flow diagram, Fig. 978.01. 

(b) Molybdovanadate reagent. — Dissolve 16.5 g NH 4 mo- 
lybdate.4H 2 in 400 mL hot H 2 0, and cool. Dissolve 0.6 g 
NH 4 metavanadate in 250 mL hot H 2 0, cool, and add 60 mL 
70% HCIO4. Gradually add molybdate soln to vanadate soln 
with stirring. Add 2 mL wetting agent, (e), and dil. to 2 L. 

(c) Perchloric acid.— AN. Add 342 mL 70% HCJO4 to 500 
mL H 2 in 1 L vol. flask. Add 1 mL wetting agent, and dil. 
to vol. 

(d) Sampler wash soln. — Add 1 mL wetting agent to 1 L 
H 2 0, and mix well. 

(e) Wetting agent. — Ultrawet 60 L (Technicon No. T01- 
0214), or equiv. 

(f) Phosphorus std solns. — (J) Stock soln. — 10 mg P 2 5 / 
mL. Dissolve 9.5880 g dried (2 hr at 105°) KH 2 P0 4 primary 
std (52.15% P 2 O s ) in H 2 0, and dil. to 500 mL with H 2 0. (2) 
Working solns.— -0.15, 0.19, 0.23, 0.27, 0.31, and 0.35 mg 
P 2 5 /mL. Using 25 mL buret, accurately measure 7.5, 9.5, 
11.5, 13.5, 15.5, and 17.5 mL stock soln into six 500 mL 
vol. flasks. Dil. each to vol. with H 2 0, and mix. (3) Working 
soln for samples <7% P 2 5 . — 2 mg P 2 5 /mL. Pipet 100 mL 
stock soln into 500 mL vol. flask, dil. to vol. with H 2 0, and 
mix. 

C. Preparation of Samples 

Prep, samples for direct available P 2 5 detn as in 960.03B(a). 
Prep, samples for total P 2 5 detn as in 957.02B(a) or (e), and 
dil. to 250 mL. 



SAMPLER II 



20T 
MIXER' 



INJECTION 
FITTING 



FLOW,ml/Min 




\ 



0.32 



AIR 



95<t] 
HEATING BATH 



40'COIL 
1.6mm ID. 



0.60 



HCIO4 



0.23 



SAMPLE 




INJECTION 
FITTING 




0-32 



AIR 



1,00 MO-V REAGENT 



0.23 DIG. SAMPLE 



20 T 
MIXER 



MODIFIED AO' 



WASTE *s- 



0.60 



Dl< 



SAMPLER 11^- 
WASTE^, 



2.00 



H20 



0.80 



FLOWCELL 




COLORIMETER RECORDER 
15 mm FLOWCELL 
420 nm FILTERS 



6 FT LENGTH 0.030 I.D.' 
TRANSMISSION TUBING 



WASTE 



POSITIONED WITH CAPILLARY SIDEARM 
ON BOTTOM 



FIG. 978.01— -Flow diagram for automated analysis for phosphorus 



AOAC Official Methods of Analysis (1990) 



Phosphorus 



15 



D. Analytical System 

(Technicon part numbers are given to aid in construction of 
manifold; equiv. coils, fittings, etc., are satisfactory.) 

Sample, air, and AN HC10 4 are combined thru injection fit- 
ting (No. 116-0489) and mixed in 20T coil (No. 157-0248). 
Stream proceeds to heating bath(s) before resample thru mod- 
ified AO fitting. Resample, air, and mo lybdo vanadate reagent 
are combined thru injection fitting (No. 116-0489). Mixing 
and color development take place in two 20T coils (No. 157- 
0248) before measurement at 420 nm. If only total P 2 5 sam- 
ples are to be analyzed, heating bath can be removed and AN 
HC10 4 soln replaced by sampler wash soln, (d). Heating bath(s) 
and acid soln are necessary only when analyzing samples for 
direct available P 2 5 or combination of direct available and 
total P 2 O s detns. 

If manifold is to be constructed following flow diagram, use 
clear std pump tubes for all air and soln flows. All fittings, 
coils, and glass transmission lines are AAII type and size. Use 
1.6 mm glass transmission tubing for all connections after pump 
to colorimeter. Construct modified AO fitting, following heat- 
ing bath, by using AO fitting, N13 stainless steel nipple con- 
nector, and 1.3 cm length of 0.035" id Tygon tubing. Insert 
N 13 nipple approx. halfway into 0.035" Tygon tubing. Insert 
tubing into side arm of AO fitting far enough so resample line 
will not pump any air. Connect Dl fitting directly to waste 
side of AO fitting; position Dl fitting with capillary side arm 
on bottom. Attach 0.6 mL/min pump tube to top arm of Dl 
fitting, and attach 1.8 m (6') of 0.030" id transmission tubing 
to bottom arm. All air segments must pass thru 0.6 mL/min 
tube, leaving continuous column of liq. in 1.8 m length of 
tubing to provide for constant back pressure on heating bath 
coil. Length of resample pump tube should be <2.5 cm from 
shoulder at entrance end. 

E. Start-Up 

Start automatic system, place all lines in resp. solns, and let 
equilibrate >30 min. Proceed as in 978. 01G. 

F. Shut-Down 

Pump water thru reagent lines >30 min. Do not remove 
HCIO4 lines from reagent until 20 min after last sample is run. 

G. Check and Calibration 

After equilibration, set colorimeter to damp 1 position and 
pump 0.15 mg P 2 5 /mL working std soln continuously thru 
system. Adjust colorimeter baseline to read 10% of full scale. 
Pump 0.35 mg P 2 5 /mL std and adjust std calibration to read 
90% of full scale. Range of 0. 15-0.35 mg P 2 O s /mL will ex- 
pand to read 10-90% of full scale. Check of bubble flow pat- 
tern will give indication of performance of system. Perfect 
bubble pattern is required to obtain optimum peak shapes. Check 
for air bubble in flowcell if noisy conditions exist. To check 
system carryover, place three 0.35 mg/mL stds, followed by 
three 0. 15 mg/mL stds, thru system. If first 0.15 mg/mL std 
following 0.35 mg/mL std is >1 chart division higher than 
other 2, carryover is indicated. If carryover occurs, check en- 
tire system for poor connections. 

H. Determination 

Pipet aliquot of sample soln (see Table 978.01) into 100 m'L 
vol. flask, dil. to vol. with H 2 0, and mix by inversion 20 
times. For sample contg <7% P 2 5 , pipet 10 ml working soln, 
(f)(3), into flask before diln. Place 0.15-0.35 mg P 2 5 /mL 
working std solns in tray in increasing order of concn, fol- 
lowed by group of samples. Analyze lowest concn std in du- 



Table 978.01 Standard Dilutions 



% p 2 5 
Expected 


Aliquot | 


mL) 




Direct Available 


Total 


Factor 


1-7 

8-16 

17-34 

^35 


50 + "spike" 
no diln 
50 
25 


25 + "spike" 

50 

25 

15 


1 

0.5 

2 for direct available 

1 .667 for total 



plicate, discarding first peak. Precede and follow each group 
of samples with std ref. curve to correct for possible drift. If 
drift between first and last set of stds is >2 chart divisions, 
repeat sample analysis. Prep, std curve by averaging peak hts 
of first and second set of stds. Plot av. peak ht of stds against 
mg P 2 5 /mL contained in each std. Read mg P 2 5 /mL for 
each sample from graph. 

% P 2 5 = mg P 2 5 /mL from graph (-0.20, if spiked) 

x F X 100 

where F = factor from Table 978.01. 

Refs.: JAOAC 61, 533(1978). 

CAS- 13 14-56-3 (phosphorus pentoxide) 



977.01 Phosphorus (Water-Soluble) 

in Fertilizers 
Preparation of Solution 
Final Action 

Place 1 g sample on 9 cm filter and wash with small portions 
H 2 until filtrate measures ca 250 mL. Add H 2 in fine stream 
directed around entire periphery of filter paper in circular path, 
ensuring that H 2 and solids are thoroly mixed with each addn. 
Let each portion pass thru filter before adding more and use 
suction if washing would not otherwise be complete within 1 
hr. If filtrate is turbid, add 1-2 mL HNO3, dil. to 250 mL, 
and mix. 

Ref.: JAOAC 60, 393,702(1977). 



962.03 Phosphorus (Water-Soluble) 

in Fertilizers 
Gravimetric Quinolinium Molybdophosphate Method 
Final Action 

Pipet aliquot contg <25 mg P 2 5 into 500 mL erlenmeyer. 
Dil., if necessary, to 50 mL, add 10 mL HN0 3 (3+1), and 
boil gently 10 min. Cool, dil. to 100 mL, and proceed as in 
962.02C(b). 



962.04 Phosphorus (Water-Soluble) 

in Fertilizers 
Alkalimetric Quinolinium Molybdophosphate Method 
Final Action 1974 

Pipet aliquot contg <30 mg P 2 5 into 500 mL erlenmeyer. 
Dil., if necessary, to 50 mL, add 10 mL HN0 3 (1 + 1), boil 
gently 10 min, cool, and proceed as in 969.02C(a), beginning 
"... add 20 mL citric acid soln . . ." 



16 



Fertilizers 



AOAC Official Methods of Analysis (1990) 



970.01 Phosphorus (Water-Soluble) 

in Fertilizers 
Spectrophotometry Molybdovanadophosphate Method 

First Action 1970 
Final Action 1974 

Adjust concn according to 958.01D(a) or (b) and proceed 
as in'958.01E. 



963.03 Phosphorus (Citrate-Insoluble) 

in Fertilizers 

First Action 1963 
Final Action 1964 

A. Reagents 

(a) Ammonium citrate soln. — Should have sp gr of 1.09 at 
20° and pH of 7.0 as detd potentiometrically. 

Dissolve 370 g cryst. citric acid in 1.5 L H 2 and nearly 
neutze by adding 345 mL NH 4 OH (28-29% NH 3 ). If concn 
of NH 3 is <28%, add correspondingly larger vol. and dissolve 
citric acid in correspondingly smaller vol. H 2 0. Cool, and check 
pH. Adjust with NH 4 OH (1+7) or citric acid soln to pH 7. 
Dil. soln, if necessary, to sp gr of 1.09 at 20°. (Vol. will be 
ca 2 L.) Keep in tightly stoppered bottles and check pH from 
time to time. If pH has changed from 7.0, readjust. 

(b) Other reagents and solns.—See 957. 02A, 958. 01B, or 
962. 02 A. 

B. Preparation of Extract 

(a) Acidulated samples, mixed fertilizers, and materials 
containing water-soluble compounds. — After removing H 2 0- 
sol. P 2 5 , 977.01, transfer filter and residue, within 1 hr, to 
200 or 250 mL flask contg 100 mL NH 4 citrate soln previously 
heated to 65°. Close flask tightly with smooth rubber stopper, 
shake vigorously until paper is reduced to pulp, and relieve 
pressure by removing stopper momentarily. Continuously ag- 
itate stoppered flask in const temp. app. at exactly 65°. (Action 
of app. should be such that dispersion of sample in citrate soln 
is continually maintained and entire inner surface of flask and 
stopper is continually bathed with soln.) 

Exactly 1 hr after adding filter and residue, remove flask 
from app. and immediately filter by suction as rapidly as pos- 
sible thru Whatman No. 5 paper, or equiv., using buchner or 
ordinary funnel with Pt or other cone. Wash with H 2 at 65° 
until vol. filtrate is ca 350 mL, allowing time for thoro drain- 
ing before adding more H 2 0. If material yields cloudy filtrate, 
wash with 5% NH 4 N0 3 soln. Prep, citrate-insol. residue for 
analysis by one of following methods: 

(7) Dry paper and contents, transfer to crucible, ignite until 
all org. matter is destroyed, and digest with 10—15 mL HO 
until all phosphate dissolves; or (2) treat wet filter and contents 
as in 957.02B(a), (c), (d), or (e). Dil. soln to 250 mL, or other 
suitable vol., mix well, and filter thru dry paper. 

(b) N onacidulated samples. — Place 1 g sample (ground to 
pass No. 40 sieve in case of Ca metaphosphate) on dry 9 cm 
paper. Without previous washing with FLO, proceed as in (a). 
If sample contains much org. matter (bone, fish, etc.), dis- 
solve residue in sol. in NH 4 citrate as in 957.02B(c), (d), or 
(e). 

C. Determination 

— Final Action 1974 

(a) Gravimetric quinolinium molybdophosphate method. — 
Treat I g sample as in 963.03B(a) or (b). Transfer aliquot of 
citrate-insol. P^O^ contg <25 mg P^0 5 and proceed as in 
962.02C. 



(b) Spectrophotometry molybdovanadophosphate method. — 
Treat 1 g sample as in 963.036(a) or (b). Adjust concn of 
citrate-insol. P 2 5 soln as in 958.01D(a) or (b) and proceed 
as in 958.01E. 

(c) Alkalimetric quinolinium molybdophosphate method. — 
Treat 1 g sample by 963.03B(a) or (b). Transfer aliquot of 
citrate-insol. P 2 5 contg <5 mL coned acid to 500 mL erlen- 
meyer. Add 20 mL 10% citric acid soln and dil. to 100 mL 
with H 2 0. Continue as in 969.02C(a), beginning "Add 60 mL 
quimociac reagent, ..." 

Refs.: JAOAC 5, 443,460(1922); 6, 384(1923); 14, 182(1931); 
19, 269(1936); 22, 254(1939); 42, 503, 512(1959); 52, 
587(1969). 

Z. Anal. Chem. 189, 243(1962). JAOAC 45, 40, 201, 
999(1962); 46, 579(1963); 47, 420(1964). 

CAS- 13 14-56-3 (phosphorus pentoxide) 



960.01 



Phosphorus (Citrate-Soluble) 
in Fertilizers 



Final Action 1960 



Subtract sum of H 2 0-sol. and citrate-insol. 
P 2 5 to obtain citrate- sol. P 2 5 . 



P 2 5 from total 



960.02 Phosphorus (Available) in Fertilizers 

Indirect Method 
Final Action 1960 

Subtract citrate-insol. P 2 5 from total P 2 5 to obtain avail- 
able P 2 5 . 

960.03 Phosphorus (Available) in Fertilizers 

Final Action 

A. Reagents 

(Caution: See safety notes on nitric acid, perchloric acid, and 
sulfuric acid.) 

(a) Nitric-perchloric acid mixture. — Add 300 mL 70% 
HCI0 4 to 700 mL HNO3. 

(b) Ternary acid mixture. — Add 20 mL H 2 S0 4 to 100 mL 
HNO3, mix, and add 40 mL 70% HC10 4 . 

(c) Modified molybdovanadate reagent. — Prep, as in 
958.016(a)' except use 250 mL 70% HCIO4 instead of 450 
mL. 

3. Preparation of Solution 

(a) Acidulated samples, mixed fertilizers, and materials 
containing water-soluble compounds. — (J) Without filtration 
of citrate digest. — Remove H 2 0-soJ. P 2 5 as in 977.01, 
collecting filtrate in 500 mL vol. flask, but do not add HNO3 
to filtrate. Treat H 2 0-insol. residue with NH 4 citrate soln as 
in 963.03B(a). Exactly 1 hr after adding filter and residue, 
remove flask from app. and transfer contents to flask contg 
H 2 0-sol. fraction. Cool to room temp, immediately, dil. to 
vol., mix thoroly, and let stand >2 hr before removing ali- 
quot. 

(2) With filtration of citrate digest.- — if desired, wash by 
gravity into 500 mL Kohlrausch flask contg 5 mL HNO3 (1 + 1), 
catching filtrate from insol. residue, 963.03B(a), in the Kohl- 
rausch flask contg H 2 0-sol. fraction, and wash residue until 
vol. soln in flask is ca 500 mL. Cool, dil. to 500 mL, and 



AOAC Official Methods of Analysis (1990) 



Nitrogen 



17 



(b) Nonacidulated samples. — Place 1 g sample (ground to 
pass No. 40 sieve in case of Ca metaphosphate) on dry 9 cm 
paper. Without previous washing with H 2 0, proceed as in (a)(7) 
or (2). If (2) is used, wash residue until vol. soln is ca 350 
ml. Cool, dil. to 500 mL, and mix. 

Refs.: JAOAC 43, 478(1960); 44, 133, 232(1961); 46, 570 
(1963); 60, 702(1977). 

C. Alkalimetric Quinolinium Molybdophosphate Method 
— Final Action 1974 

Treat 1 g sample by appropriate modification of 960. 03B. 
Transfer aliquot contg <30 mg P 2 5 and <10 mL NH 4 citrate 
soln, 963.03A(a), to 500 mL erlenmeyer. DiL, if necessary, 
to 50 mL, add 10 mL HN0 3 (1 + 1), and boil gently 10 min. 
Cool, dil. to 100 mL, and continue as in 969.02C(a), begin- 
ning "Add 60 mL quimociac reagent, ..." 

Ref.: JAOAC 52, 587(1969). 

D. Spectrophotometry Molybdovanadophosphate Method 
—Final Action 1961 

(Not applicable to materials yielding colored solns or solns 
contg ions other than orthophosphate which form colored com- 
plexes with molybdovanadate. Not recommended for basic 
slag.) 

Prep, std curve as in 958.01 C, using photometer, 958. 01 A. 

Pipet, into 100 mL vol. flasks, 5 mL aliquots std phosphate 
solns contg 2 and 3.5 mg P 2 5 /aliquot, 958.01B(b), resp., add 
2 mL 70% HC10 4 , and develop color as in 958.01C. Adjust 
instrument to zero A for 2 mg std and det. A of 3.5 mg std. 
(A of latter must be practically identical with corresponding 
value on std curve.) 

Prep, sample as in 960. 03B. 

(a) Samples containing up to 5% P 2 O s . — Pipet 10 mL sam- 
ple soln into 125 mL erlenmeyer, and treat by one of following 
methods (Caution: See safety notes on wet oxidation, nitric 
acid, and perchloric acid): 

(J) Add 5 mL 20% NaC10 3 soln and 10 mL HN0 3 -HC10 4 
mixt., 960.03A(a). Boil gently until greenish-yellow color dis- 
appears (ca 20 min), cool, and add 2 mL HC1. After vigorous 
reaction subsides, evap. to fumes of HCIO4, and fume 2 min. 

(2) Add 5 mL ternary acid mixt., 960.03A(b), swirl, boil 
gently 15 min, and digest at 150-200° until clear white salt 
or colorless soln remains. Evap. to white fumes and continue 
heating 5 min. 

Cool, add 15 mL H 2 0, and boil 5 min. Transfer to 100 mL 
vol. flask, dil. to 50 mL, swirl, and cool to room temp. Add 
5 mL std phosphate soln contg 2 mg P 2 5 and 20 mL modified 
molybdovanadate soln, 960.03A(c). Dil. to 100 mL, and con- 
tinue as in 958.01E. 

(b) Samples containing more than 5% P20 5 . — Dil. soln to 
such vol. that 5-10 mL aliquot contains 2-5 mg P?0 5 . Digest 
as in (a)(7) or (2). Without adding std phosphate soln, con- 
tinue as in (a). 

Ref.: JAOAC 44, 233(1961). 

E. Gravimetric Quinolinium Molybdophosphate Method 
—First Action 1963 

—Final Action 1964 

(a) Solns containing no organic phosphorus. — Prep, sam- 
ple as in 960.03B. Pipet, into 500 mL erlenmeyer, aliquot contg 
<25 mg P 2 5 and <10 mL original NH 4 citrate soln. DiL, if 
necessary, to ca 50 mL, add 10 mL H.NO3 (1 + 1), and boil 
gently 10 min. Cool, dil. to 150 mL, and proceed as in 
962.02C(a) or (b). 

(b) Solns containing organic phosphorus . — (Caution: See 



safety notes on wet oxidation, nitric acid, and perchloric acid.) 
Select aliquot as in (a). Add 10 mL 20% NaC10 3 and 10 mL 
HNO3-HCIO4 mixt., 960.03A(a). Boil vigorously until green- 
ish-yellow color disappears (usually ca 30 min), cool, and add 
2 mL HO. After vigorous reaction subsides, evap. to white 
fumes, and continue heating 5 min. Cool, and proceed as in 
962.02C(a) or (b). 

Refs.: JAOAC 46, 570(1963); 47, 420(1964). 



920.01 



NITROGEN 

Nitrates in Fertilizers 

Detection Method 

Final Action 



Mix 5 g sample with 25 mL hot H 2 0, and filter. To 1 vol. 
of this soln add 2 vols H 2 S0 4 , free from HN0 3 and oxides of 
N, and let cool. Add few drops coned FeS0 4 soln in such 
manner that fluids do not mix. If nitrates are present, junction 
at first shows purple, afterwards brown, or if only minute amt 
is present, reddish color. To another portion of soln add 1 mL 
1% NaN0 3 soln and test as before to det. whether enough H 2 S0 4 
was added in first test. 



955.04 Nitrogen (Total) in Fertilizers 

Kjeldah! Method 
Final Action 

(Provide adequate ventilation in laboratory and do not permit 
accumulation of exposed Hg.) 

A. Reagents 

(a) Sulfuric acid.— -93-98% H 2 S0 4 , N-free. 

(b) Mercuric oxide or metallic mercury. — HgO or Hg, re- 
agent grade, N-free. 

(c) Potassium sulfate (or anhydrous sodium sulfate). — Re- 
agent grade, N-free. 

(d) Salicylic acid. — Reagent grade, N-free. 

(e) Sulfide or thiosulfate soln. — Dissolve 40 g com. K 2 S in 
1 L H 2 0. (Soln of 40 g Na 2 S or 80 g Na 2 S 2 3 .5H 2 in 1 L 
may be used.) 

(f) Sodium hydroxide .—(Caution: See safety notes on so- 
dium and potassium hydroxide.) Pellets or soln, nitrate-free. 
For soln, dissolve ca 450 g solid NaOH in H 2 0, cool, and dil. 
to 1 L. (Sp gr of soln should be >'L36.) 

(g) Zinc granules. — Reagent grade, 
(h) Zinc dust. — Impalpable powder. 

(i) Methyl red indicator. — Dissolve 1 g Me red in 200 mL 
alcohol. 

(j) Hydrochloric or sulfuric acid std soln. — 0.5vV, or 0. \N 
when amt of N is small. Prep, as in 936.15 or 890. 01 A. 

(k) Sodium hydroxide std soln. — 0.17V (or other specified 
concn). Prep, as in 936.16. 

Stdze each std soln with primary std (see chapter on standard 
solutions) and check one against the other. Test reagents be- 
fore use by blank detn with 2 g sugar, which ensures partial 
reduction of any nitrates present. 

Caution: Use freshly opened H 2 S0 4 or add dry P 2 5 to avoid 
hydrolysis of nitriles and cyanates. Ratio of salt to acid (wt:vol.) 
should be ca 1:1 at end of digestion for proper temp, control. 
Digestion may be incomplete at lower ratio; N may be lost at 
higher ratio. Each g fat consumes 10 mL H 2 S0 4 , and each g 
carbohydrate 4 mL H 2 S0 4 during digestion. 



18 



Fertilizers 



AOAC Official Methods of Analysis (1990) 



B. Apparatus 

(a) For digestion. — Use Kjeldahl flasks of hard, moder- 
ately thick, well-annealed glass with total capacity ca 500-800 
mL. Conduct digestion over heating device adjusted to bring 
250 mL H 2 at 25° to rolling boil in ca 5 min or other time 
as specified in method. To test heaters, preheat 10 min if gas 
or 30 min if elec. Add 3-4 boiling chips to prevent super- 
heating. 

(b) For distillation. — Use 500-800 mL Kjeldahl or other 
suitable flask, fitted with rubber stopper thru which passes lower 
end of efficient scrubber bulb or trap to prevent mech. car- 
ryover of NaOH during distn. Connect upper end of bulb tube 
to condenser tube by rubber tubing. Trap outlet of condenser 
in such way as to ensure complete absorption of NH 3 distd 
over into acid in receiver. 

C. improved Kjeldahl Method for Nitrate-Free Samples 

(Caution: See safety notes on sulfuric acid, sodium hydrox- 
ides, and mercury.) 

Place weighed sample (0.7-2.2 g) in digestion flask. Add 
0.7 g HgO or 0.65 g metallic Hg, 15 g powd K 2 S0 4 or anhyd. 
Na 2 S0 4 , and 25 mL H 2 S0 4 . If sample >2.2 g is used, increase 
H 2 S0 4 by 10 mL for each g sample. Place flask in inclined 
position and heat gently until frothing ceases (if necessary, add 
small amt of paraffin to reduce frothing); boil briskly until soln 
clears and then >30 min longer (2 hr for samples contg org. 
material) . 

Cool, add ca 200 mL H 2 0, cool <25°, add 25 mL of the 
sulfide or thiosulfate soln, and mix to ppt Hg. Add few Zn 
granules to prevent bumping, tilt flask, and add layer of NaOH 
without agitation. (For each 10 mL H 2 S0 4 used, or its equiv. 
in dild H 2 S0 4 , add 15 g solid NaOH or enough soln to make 
contents strongly alk.) (Thiosulfate or sulfide soln may be mixed 
with the NaOH soln before addn to flask.) Immediately con- 
nect flask to distg bulb on condenser, and, with tip of con- 
denser immersed in std acid and 5-7 drops indicator in re- 
ceiver, rotate flask to mix contents thoroly; then heat until all 
NH 3 has distd (>150 mL distillate). Remove receiver, wash 
tip of condenser, and titr. excess std acid in distillate with std 
NaOH soln. Correct for blank detn on reagents. 

% N = [(mL std acid x normality acid) - (mL std NaOH 
x normality NaOH)] x].4007/g sample 

Ref: JAOAC 38, 56(1955). 

D. Improved Kjeldahl Method for Nitrate-Containing Samples 

(Not applicable to liqs or to materials with high C1:N0 3 ratio. 
Caution: See safety notes on sulfuric acid and mercury.) 

Place weighed sample (0.7-2.2 g) in digestion flask. Add 
40 mL H 2 S0 4 contg 2 g salicylic acid. Shake until thoroly 
mixed and Jet stand, with occasional shaking, >30 min; then 
add (/) 5 g Na 2 S 2 3 .5H 2 or (2) 2 g Zn dust (as impalpable 
powder, not granulated Zn or filings). Shake and let stand 5 
min; then heat over low flame until frothing ceases. Turn off 
heat, add 0.7 g HgO (or 0.65 g metallic Hg) and 15 g powd 
K 2 S0 4 (or anhyd. Na 2 S0 4 ), and boil briskly until soln clears, 
then >30 min longer (2 hr for samples contg org. material). 

Proceed as in second par. of 955. 04C. 

Ref.: JAOAC 51, 446(1968). 
CAS-7727-37-9 (nitrogen) 



970.02 Nitrogen (Tota!) in Fertilizers 

Comprehensive Nitrogen Method 

First Action 1970 
Final Action 1975 

(Applicable to all fertilizer samples.) 
(Caution: See safety notes on sulfuric acid and mercury salts.) 

A. Reagents 

(a) Chromium metal. — 100 mesh, low N (Fisher Scientific 
Co. No. C-318 or Sargent- Welch Scientific Co. No. C1 1432 
is satisfactory). 

(b) Alundum. — Boiling stones. 8-14 mesh (Thomas Sci- 
entific No. 1590-D18, or equiv.). 

(c) Dilute sulfuric acid. — Slowly add 625 mL H 2 S0 4 to 300 
mL H 2 0. Dil. to ca 1 L and mix. After cooling, dil. to 1 L 
with H 2 and mix. Avoid absorption of NH 3 from air during 
prepn, particularly if stream of air is used for mixing. 

(d) Sodium thiosulfate or potassium sulfide soln.—] 60 g 
Na 2 S 2 3 .5H 2 0/L or 80 g K 2 S/L. 

For other reagents, see 920. 02A. 

B. Determination 

Place 0.2-2.0 g sample contg <60 mg nitrate N in 500- 
800 mL Kjeldahl flask and add 1.2 g Cr powder. Add 35 mL 
H 2 or, with liqs, amt to make total vol. 35 mL. Let stand 
10 min with occasional gentle swirling to dissolve all nitrate 
salts. Add 7 mL HC1 and let stand >30 sec but <10 min. 

Place flask on preheated burner with heat input set at 7.0- 
7.5 min boil test, 920.02B(a). After heating 3.5 min, remove 
from heat and let cool. 

Add 22 g K 2 S0 4 , 1.0 g HgO, and few granules Alundum. 
Add 40 mL dil. H 2 S0 4 , (c). (If adequate ventilation is avail- 
able, 25 mL H 2 S0 4 may be added instead of dil. H 2 S0 4 . If 
org. matter which consumes large amt of acid exceeds 1.0 g, 
add addnl 1.0 mL H?S0 4 for each 0.1 g org. matter in excess 
of 1.0 g.) 

Place flask on burners set at 5 min boil test. (Pre-heated 
burners reduce foaming with most samples. Reduce heat input 
if foam fills > 2 / 3 of bulb of flask. Use variable heat input until 
this phase is past.) Heat at 5 min boil test until dense white 
fumes of H 2 S0 4 clear bulb of flask. Digestion is now complete 
for samples contg ammoniacal, nitrate, and urea N. For other 
samples, swirl flask gently and continue digestion 60 min more. 

Proceed as in 955.04C, second par., substituting 970.02A(d) 
for 920.02 A(e). 

Refs.: JAOAC 53, 450(1970); 57, 10(1974); 68, 441(1978). 

CAS-7727-37-9 (nitrogen) 



978.02 Nitrogen (Total) in Fertilizers 

Modified Comprehensive Nitrogen Method 

First Action 1978 
Final Action 1984 

(Applicable to all fertilizer samples) 

A. Reagents 

See 920.02A(a), (c), (f), (i), (j), (k), 970.02A(a), (b), and 
in addn: 

Copper sulfate pentahydrate (or anhydrous copper sul- 
fate). — Reagent grade, N-free. 



AOAC Official Methods of Analysis (1990) 



Nitrogen 



19 



B. Determination 

{Caution: See safety notes on wet oxidation, sulfuric acid, and 
sodium hydroxide.) 

Proceed as in 970. 02B, par. I and 2, using 0.2-1.6 g sam- 
ple. For samples contg orgs other than urea or urea-form, use 
>0.5 g sample. 

Add 15 g K 2 S0 4 or 12 g anhyd. Na 2 S0 4 , 0.4 g anhyd. CuS0 4 
or 0.6 g CuS0 4 .5H 2 0, and ca 0.8 g Alundum granules. Add 
37 inL H 2 S0 4 (1 + 1). (If adequate ventilation is available, 20 
mL H 2 S0 4 may be added instead of H 2 S0 4 (1 + 1). If org. mat- 
ter other than urea exceeds 1.0 g, add addnl 1.0 mL H 2 S0 4 
for each 0.1 g fat or 0.2 g other org. matter in excess of 1 .0 

g.) 

Proceed as in 970. 02B, par. 4, substituting 75 min for 60 
min in last sentence. 

Cool flask until it can be handled without gloves, and add 
ca 250 mL H 2 0. Swirl to dissolve contents, and cool <25°. 
Add ca 0.8 g Alundum granules to minimize bumping, tilt 
flask, and add layer of NaOH without agitation. (For each 10 
ml H 2 S0 4 used, or its equiv. in H 2 S0 4 (1 + 1), add 15 g solid 
NaOH or enough so In to make contents strongly alk.) Proceed 
as in 955. 04C, par. 2, beginning "Immediately connect flask 
to distg bulb ..." 

Ref.: JAOAC 61, 299(1978). 

CAS-7727-37-9 (nitrogen) 



970.03 Nitrogen (Total) in Fertilizers 

Raney Powder Method 

First Action 1970 
Final Action 1975 

(Applicable to all fertilizer samples except "nitric phosphates" 

contg nonsulfate S. Caution: See safety notes on sulfuric acid 

and mercury salts.) 

A. Reagents 

(a) Raney catalyst powder No. 2813.-50% Ni, 50% Al 
(W. R. Grace & Co., Davison Chemical Division, 10 E Bal- 
timore St, PO Box 2117, Baltimore, MD 21203-2117). Cau- 
tion: Raney catalyst powders react slowly in H 2 or moist air 
to form alumina; avoid prolonged contact with air or moisture 
during storage or use. 

(b) Sulfuric acid-potassium sulfate soln. — Slowly add 200 
mL H 2 S0 4 to 625 mL H 2 and mix. Without cooling, add 
106.7 g K 2 S0 4 and continue stirring until all salt dissolves. 
Dil. to ca 1 L and mix. Cool, dil. to 1 L with H 2 0, and mix. 
Avoid absorption of NH 3 from air during prepn particularly if 
stream of air is used for mixing. 

For other reagents, see 920. 02 A. 

B. Determination 

Place 0.2-2.0 g sample contg <42 mg nitrate N in 500- 
800 mL Kjeldahl flask (800 mL flask is preferred with samples 
which foam considerably, especially orgs). Add 1.7 g Raney 
catalyst powder, 3 drops tributyl citrate, and 150 mL H 2 S0 4 - 
K 2 S0 4 soln. If org. matter exceeds 0.6 g, add addnl 2.5 mL 
of this soln for each 0. 1 g org. matter in excess of 0.6 g. 

Swirl to mix sample with acid and place flask on cold burner. 
If burner has been in use, turn off completely >10 min before 
placing flask on burner. After flask is on burner, set heat input 
to 5 min boil test. When sample starts boiling, reduce heat to 
pass 10 min boil test. After 10 min, raise flask to vertical po- 



sition and add 0.7 g HgO and 15 g K 2 S0 4 . (Contents of Kel- 
Pak No. 5 (Curtin Matheson Scientific, Inc.) without plastic 
container may be used.) Replace flask in inclined position and 
increase heat to 4-5 min boil test. (Reduce heat input if foam 
fills >: 2 /3 of bulb of flask. Use variable heat input until this 
phase is past.) Heat at 4-5 min boil test until dense white 
fumes of H 2 S0 4 clear bulb of flask. Digestion is now complete 
for samples contg only ammoniacal, nitrate, and urea N. For 
other samples, swirl flask gently and continue digestion addnl 
30 min. 

Proceed as in 955. 04C, second par. If 800 mL Kjeldahl flasks 
have been used, add 300 instead of 200 mL H 2 0. 

Refs.: JAOAC 53, 450(1970); 57, 10(1974). 

CAS-7727-37-9 (nitrogen) 



920.03 Nitrogen (Ammoniacal) 

in Fertilizers 
Magnesium Oxide Method 
Final Action 

(Not applicable in presence of urea) 

Place 0.7-3.5 g, according to NH 3 content of sample, in 
distn flask with ca 200 mL H 2 and >:2 g carbonate-free MgO. 
Connect flask to condenser by Kjeldahl connecting bulb, distil 
100 mL liq. into measured amt std acid, 920.02A(j), and titr. 
with std NaOH soln, 920.02A(k), using Me red, 920.02A(i). 



920.04* Nitrogen (Ammoniacal) 

in Fertilizers 
Formaldehyde Titration Method 
Surplus 1970 

(Applicable to NH 4 N0 3 and (NH 4 ) 2 S0 4 ) 
See 2.058, 11th ed. 



920.05* Nitrogen (Ammoniacal and Nitrate) 
in Fertilizers 
Ferrous Sulfate-Zinc-Soda Method 

Final Action 1965 
Surplus 1970 

(Not applicable in presence of org. matter, Ca cyanamide, and 

urea) 

See 2.059, 11th ed. 



892.01 Nitrogen (Ammoniacal and Nitrate) 
in Fertilizers 
Devarda Method 
Final Action 

(Not applicable in presence of org. matter, Ca cyanamide, and 

urea) 

Place 0.35 or 0.5 g sample in 600-700 mL flask and add 
300 mL H 2 0, 3 g Devarda alloy (Cu 50, Al 45, Zn 5), and 



20 



Fertilizers 



AOAC Official Methods of Analysis (1990) 



5 mL NaOH soln (42% by wt), pouring latter down side of 
flask so that it does not mix at once with contents. By means 
of Davisson (J. ind. Eng. Chem. 11, 465(1919)) or other suit- 
able scrubbing bulb that will prevent passing over of any spray, 
connect with condenser, tip of which always extends beneath 
surface of std acid in receiving flask. Mix contents of distg 
flask by rotating. Heat slowly at first and then at rate to yield 
250 mL distillate in 1 hr. Collect distillate in measured amt 
std acid, 920.02A(j), and titr. with std NaOH soln, 920.02A(k), 
using Me red, 920.02 A(i). 

Refs.: Chem. Ztg. 16, 1952(1892). JAOAC 6, 391(1923); 15, 
267(1932). 



930.01 Nitrogen (Nitrate) in Fertilizers 

Robertson Method 
Final Action 

(Applicable in presence of Ca cyan amide and urea. Caution: 
See safety notes on sulfuric acid and mercury.) 

(a) Det. total N as in 955.04D, 970.02B, or 970.03B. 

(b) Det. H 2 Oinsol. N as in 945.01, but use 2.5 g sample. 
Dil. filtrate to 250 mL. 

(c) Place 50 mL portion filtrate in 500 mL Kjeldahl flask 
and add 2 g FeS0 4 .H 2 and 20 mL H 2 S0 4 . (If total N is >5%, 
use 5 g FeS0 4 .7H 2 0.) Digest over hot flame until all H 2 is 
evapd and white fumes appear, and continue digestion at least 
10 min to drive off nitrate N. If severe bumping occurs, add 
10-15 glass beads. Add 0.65 g Hg, or 0.7 g HgO, and digest 
until all org. matter is oxidized. Cool, dil., add the K 2 S soln, 
and complete detn as in 955. 04C. Before distn, add pinch of 
mixt. of Zn dust and granular "20-mesh" Zn to each flask to 
prevent bumping. 

Total N (a) - H 2 0-insol. N (b) = H 2 0-sol. N. 
H 2 0-sol. N - N obtained in (c) - nitrate N. 

Refs.: JAOAC 13, 208(1930); 15, 267(1932); 56, 392(1973). 



930.02 Nitrogen (Nitrate) in Fertilizers 

Jones Modification of Robertson Method 
Final Action 

(Applicable when H 2 0-sol. N need not be detd.) 

{Caution: See safety notes on sulfuric acid and mercury.) 

Weigh 0.5 g sample into Kjeldahl flask, add 50 mL H 2 0, 
and rotate gently. Add 2 g FeS0 4 .7H 2 and rotate. Add 20 
mL H 2 S0 4 . Digest over hot flame. When H 2 evaps and white 
fumes appear, add 0.65 g Hg and proceed as in 955. 04C. 

Total N — N thus found = nitrate N 

Refs.: JAOAC 13, 208(1930); 15, 267(1932). 



945.01 Nitrogen (Water-Insoluble) 

in Fertilizers 

Method I 
Final Action 

(See 955.058(a) and (b) for urea-formaldehyde or mixts contg 
such compds.) 

Place 1 or 1.4 g sample in 50 mL beaker, wet with alcohol, 
add 20 mL H 2 0, and let stand 15 min, stirring occasionally. 
Transfer supernate to 1 1 cm Whatman No. 2 paper in 60° long- 
stem funnel 60 mm diam., and wash residue 4 or 5 times by 
decanting with H 2 at room temp. (20-25°). Finally transfer 
all residue to filter and complete washing until filtrate mea- 
sures 250 mL. Det. N in residue as in 955. 04C. 



970.04 Nitrogen (Water-Insoluble) 

in Fertilizers 
Method II 
First Action 1974 



10 mm id, 12 mm od, con- 



A. Apparatus 

Extraction tube. — Glass, 250 X 
stricted to 3-4 mm at one end. 

B. Determination 

Weigh 3.0 g unground mixed sample and place in extn tube 
contg small glass wool plug. Place addnl glass wool pad on 
top of sample. Connect 250 or 500 mL separator to column 
with 75 mm piece of rubber tubing. Close stopcock of sepa- 
rator and add 250 mL deionized H 2 0. Open stopcock and let 
quick rush of H 2 pass thru column. After initial rush of H 2 0, 
close stopcock. Adjust flow thru stopcock to ca 2 mL/min. 
Squeeze rubber connection to bring level of H 2 ca 25 mm 
above column bed. System then operates as constant-head 
feeder. 

After H 2 wash is complete, disconnect column from rub- 
ber tubing. Invert column over Kjeldahl flask and force con- 
tents into flask with aid of pressure bulb. Wash traces of sam- 
ple from tube into Kjeldahl flask and wash sample from walls 
of digestion flask with min. H 2 0. Det. N in residue as in 970.02 
or 970.03. 

Refs.: JAOAC 53, 808(1970); 56, 853(1973). 



930.03* Nitrogen Activity in Fertilizers 

Removal of Water-Soluble Nitrogen 

Final Action 
Surplus 1967 

(a) Mixed fertilizers. — See 2.058, 10th ed. 

(b) Raw materials. — See 2.058, 10th ed. 



935.01* Nitrogen (Water-Insoluble) 

in Cyanamide 

Final Action 
Surplus 1970 



920.06* Nitrogen Activity in Fertilizers 

Water-Insoluble Organic Nitrogen 
Soluble in Neutral Permanganate 

Final Action 
Surplus 1987 



See 2.063, 11th ed. 



See 2.059, 10th ed. 



AOAC Official Methods of Analysis (1990) 



Nitrogen 



21 



920.07* Nitrogen Activity in Fertilizers 

Water-Insoluble Organic Nitrogen 
Distilled from Alkaline Permanganate 

Final Action 
Surplus 1987 

See 2.060-2.061, 10th cd. 



955.05 Nitrogen Activity Index (Al) 

of Urea-Formaldehyde Fertilizers 
Final Action 1965 

(Applicable to urea-formaldehyde compds and mixts contg such 
compds) 

A. Reagent 

Phosphate buffer soln.—pH 7.5. Dissolve 14.3 g KH 2 P0 4 
and 91.0 g K 2 HP0 4 in H 2 and dil. to 1 L. Dil. 100 mL of 
this soln to 1 L. 

B. Determination 

(a) Crush sample (do not grind) to pass No. 20 sieve. 

(b) Det. cold H 2 0-insol. N (WIN) as in 945.01, keeping 
temp, at 25±2°. Stir at 5 min intervals during 15 min standing. 

(c) Det. hot H 2 0-insol. N (HWIN) in phosphate buffer soln 
as follows: Place accurately weighed sample contg 0.1200 g 
WIN in 200 mL tall-form beaker. Add ca 0.5 g CaC0 3 to mixed 
fertilizers contg urea-HCHO compds. From supply of boiling 
buffer soln, add 100 mL from graduate to sample, stir, cover, 
and immerse promptly in boiling H 2 bath so that liq. in beaker 
is below H 2 level in bath. Maintain bath at 98-100°, checked 
with thermometer, and stir at 10 min intervals. After exactly 
30 min, remove beaker from bath and filter promptly thru 15 
cm Whatman No. 12 fluted paper. If filtration takes >4 min, 
discard detn. Repeat detn, stirring in 1 g Celite filter-aid just 
before removing beaker from bath, and filter. 

Wash insol. residue completely onto paper with boiling H 2 
and continue washing until total vol. used is 100 mL. Com- 
plete washing before filtrate becomes cloudy or its temp, drops 
to <60°. Det. total N (HWIN) in wet paper and residue as in 
955.04C, using 35 mL H 2 S0 4 when CaC0 3 has been added. 

Activity index (Al) = (%WIN - %HWIN) x 100/% WIN 

Refs: JAOAC 38, 436(1955); 44, 245(1961). 



959.03 Urea in Fertilizers 

Urease Method 

First Action 1959 
Final Action 1960 

A. Reagent 

Neutral urease soln. — Use fresh com. 1% urease soln, or 
dissolve 1 g urease powder in 100 mL H 2 0, or shake 1 g jack 
bean meal with 100 mL H 2 5 min. Transfer 10 mL soln to 
250 mL erlenmeyer, dil. with 50 mL H 2 0, and add 4 drops 
Me purple (available from Fisher Scientific Co.; No. So-I-9). 
Titr. with 0.17V HO to reddish purple; then back-titr. to green 
with 0. lA r NaOH. From difference in mL, calc. vol. 0. IN HC1 
required to neutze remainder of soln (usually ca 2.5 mL/100 
mL), add this amount of acid, and shake well. 

Verify enzyme activity of urease source periodically. Dis- 
card any source which does not produce soln capable of hy- 
drolyzing 0.1 g urea/20 mL soln. 



B. Determination 

Weigh 1-10±0.01 g sample (<1.0 g urea) and transfer to 
15 cm Whatman No. 12 fluted filter paper. Leach with ca 300 
mL H 2 into 500 mL vol. flask. Add 75-100 mL satd Ba(OH) 2 
soln to ppt phosphates. Let settle and test for complete pptn 
with few drops satd Ba(OH) 2 soln. Add 20 mL 10% Na 2 C0 3 
soln to ppt excess Ba and any sol. Ca salts. Let settle and test 
for complete pptn. Dil. to vol., mix, and filter thru 15 cm 
Whatman No. 12 fluted paper. Transfer 50 mL aliquot to 200 
or 250 mL erlenmeyer and add 1-2 drops of Me purple. Acid- 
ify with 2N HC1 and add 2-3 drops excess. Neutze soln with 
0AN NaOH to first change in color of indicator. Add 20 mL 
neutral urease soln, close flask with rubber stopper, and let 
stand 1 hr at 20-25°. Cool flask in ice-H 2 slurry and titr. at 
once with 0AN HC1 to full purple; then add ca 5 mL excess. 
Record total vol. added. Back-titr. excess HC1 with 0AN NaOH 
to neut. end point. 

% Urea = [(mLO AN HC1 - mL 0. \N NaOH) 

X 0.3003]/g sample 

Refs.: Ind. Eng. Chem. Anal. Ed. 7, 259(1935). JAOAC 41, 
637(1958); 42, 494(1959); 43, 123(1960). 

CAS-57-13-6 (urea) 



983.01 Urea and Methyleneureas 

(Water-Soluble) in Fertilizers 
Liquid Chromatographic Method 

First Action 1983 
Final Action 1984 

A. Principle 

Sample is ground to pass 40 mesh sieve, extd with H 2 0, 
and filtered. Urea, methylenediurea (MDU), and dimethyl- 
enetriurea (DMTU) are detd by liq. chromatgy using external 
stds and refractive index detection. 

B. Apparatus 

(a) Liquid chromato graph. — With refractive index detector 
and pump capable of delivering mobile phase at 2 mL/min at 
pressures up to 2000 psig. Operating conditions: flow rate 1.0 
mL/min (1500 psi); attenuator 8x; ambient temp.; injection 
vol. 10 \xL. Sample injector with fixed sample loop preferred. 

(b) Chromatographic column. — Partisil 5 ODS-3, 4.6 mm 
id x 25 cm (Whatman, Inc.; other manufacturers' small par- 
ticle reverse phase columns may be substituted with adjust- 
ments in operating conditions). 

(c) Strip chart recorder. — Range to match output of de- 
tector. 

C. Reagents 

(a) Mobile phase. — LC grade H 2 0. 

(b) Purified methylenediurea (MDU) and dime thy lenetri- 
urea (DMTU).- — Ext 50 g N-only ureaformaldehyde (UF) fer- 
tilizer with acetone 8 h on soxhlet extractor. Select UF fertil- 
izer with high MDU /DMTU -to-urea ratio. Remove thimble 
from extractor, let air-dry, and collect residue. Mix 30 g ace- 
tone-washed residue in 300 mL H 2 and filter or centrf . Inject 
100 mL supernate onto Waters Associates PrepPak 500 C-18 
cartridge (5.7x30 cm) in preparative liq. chromatograph (Waters 
Associates Inc. Prep-500, orequiv.) at ambient temp, and with 
H 2 mobile phase at 150 mL/min. Collect top third of MDU 
and DMTU peaks. Evap. collected fractions to dryness in hood, 
using heat lamps. Dry using vac. over P 2 5 . Confirm identity 
using anal. liq. chromatogy and elemental analysis: mp of pu- 



22 



Fertilizers 



AOAC Official Methods of Analysis (1990) 



rified material, detd in Pyrex, should be 205-207°d for MDU 
and 231~232°d for DMTU. 

(c) External std solns. — (A) Accurately weigh ca 1.0 g each 
of urea (Baker Analyzed Reagent) and purified MDU, transfer 
both weighed compds to same 100 mL vol. flask, and dil. to 
vol. with H 2 0. (B) Accurately weigh 0.0125, 0.025, 0.050, 
and 0.10 g purified DMTU into sep. 50 mL vol. flasks. (C) 
Pipet 2, 5, 10, and 15 mL of mixed urea/MDU stds (A) into 
the vol. flasks from ( B), resp. Dil. to ca 40 mL with H 2 and 
warm as necessary to dissolve DMTU. Cool to room temp. 
and dil. to vol. Approx. std contents - (7) 0.25 mg DMTU 
+ 0.4 mg urea/MDU per mL; (2) 0.50 mg DMTU + 1.0 mg 
urea/MDU per mL; (3) 1 .00 mg DMTU + 2.0 mg urea/MDU 
per mL; (4) 2.00 mg DMTU + 3.0 mg urea/MDU per mL. 

D. Preparation of Sample 

Grind sample to pass 40 mesh sieve. Accurately weigh 2.000 
g well mixed ground sample into 200 mL vol. flask. Add 150 
mL distd or deionized H 2 0, place on wrist-action shaker 20 
min, and dil. to vol. with H 2 0. Using glass fiber paper, filter 
portion into 4 mL vial. Filter again thru 0.45 jim filter before 
injection. 

E. Determination and Calculations 

Inject 10 |xL of each mixed std until peak hts agree ±2%. 
Inject 10 [xL sample. Repeat stds after all samples have been 
injected. Std peak hts should agree within 3% of initial std 
peak hts. Average peak hts for each component and plot mg/ 
mL vs peak hts. 

% Urea N = rag/mL (from graph) x 9.33/g sample 

% MDU N = mg/mL (from graph) X 8.484/g sample 

% DMTU N - mg/mL (from graph) x 8.236/g sample 

Ref.: JAOAC 66, 769(1983). 

CAS-57-13-6 (urea) 

CAS- 1 3547- 1 7-6 (methy Jenediurea) 



988.01 Triamino-s-Triazine 

in Fertilizer Mixes 
Liquid Chromatographic Method 
First Action 1988 



C. Reagents 

(a) Sodium phosphate. — Anhyd., dibasic. Na 2 HP0 4 , re- 
agent grade or equiv. 

(b) Diethy lamine. — Reagent grade or equiv. 

(c) Phosphoric acid. — Reagent grade or equiv. 

(d) Water. — Deionized or distd. 

(e) Buffer soln.— pH 4.0. 

(f) Mobile phase. — Deionized H 2 contg 1% (w/v) anhyd. 
Na 2 HP0 4 and 1 mL diethylamine/L. Adjust to pH 4 with H 3 P0 4 . 

(g) Triamino-s-triazine std solns . — (I) Stock std so In . — 5 00 
mg/L (ppm). Accurately weigh 50.0 mg triamino-s-triazine 
ref. std (Melamine Chemicals, Inc., PO Box 748, Donaldson- 
ville, LA 70346) into 100 mL vol. flask. Dissolve in and dil. 
to vol. with deionized H a O. (2) Working std solns. — 50, 125, 
and 250 mg/L. Pipet 10, 25, and 50 mL stock std soln into 
sep. 100 mL vol. flasks and dil. to vol. with deionized H 2 0. 
Use as calibration stds. 

D. Preparation of Sample 

Grind >225 g sample (triamino-s-triazine granules or dry- 
mix blends with other fertilizers) to pass No. 40 sieve, mix 
thoroly, and store in tightly stoppered bottle. 

Accurately weigh 5-8 g well mixed, ground sample and 
transfer to 2 L vol. flask. Dil. to vol. with deionized H 2 and 
stir 2 h using stir bar and mag. stirrer. Filter portion for analy- 
sis thru 1 (xm glass fiber filter. Pipet 1 mL filtrate into 100 
mL vol. flask and dil. to vol. with deionized H 2 0. 

E. Determination 

Equilibrate column with mobile phase for 30-60 min. Inject 
20 (xL std soln until peak hts agree ±2%. Inject 20 |ulL sample 
with attenuation set to give largest possible on-scale peaks. 
Reinject std after every 10th sample to verify calibration and 
ensure accurate quantitation. 

F. Calculations 

Calc. amt triamino-s-triazine as follows: 

Triamino-s-triazine, % - {PH/PH') X |C/(5 x W)] x 100 

where PH and PH' - peak hts for sample and std, resp.; 
C = concn of std, ppm; and W = sample wt, g. 

Ref.: J AOAC 71, 611(1988). 

CAS- 108-78-1 (l,3,5-triazine-2,4,6-triamine; melamine) 



A. Principle 

Ground sample is extd with H 2 and filtered. Triamino-s- 
triazine is detd by liq. chromatgy using external std and UV 
detection at 254 nm. 

B. Apparatus 

(a) Liquid chromatograph. — With UV detection at 254 nm. 
Operating conditions: flow rate 1.0 mL/min (1200 psi); col- 
umn temp, ambient; chart speed 0.5 cm/min; injection vol. 20 
(xL; sample injector with fixed sample loop preferred. Pump 
LC mobile phase thru column until system is equilibrated. Al- 
low 10 min run time for each injection. Retention time for 
triamino-s-triazine is 4-5 min. Re-equilibrate baseline before 
each injection. 

(b) LC column.— LiChrosorb RP-18, 25 cm x 4.5 mm. (Use 
this type column; chemistry of triamino-s-triazine requires use 
of polar solv. system.) 

(c) Strip chart recorder. — To match output of detector. 

(d) pH meter. — Sensitivity 0.01. Stdze with pH 4 buffer 
soln. 

(e) Filters. — 2.4 cm glass fiber (Whatman 934-H or equiv.). 



960.04 Biuret in Fertilizers 

Spectrophotometric Method 

First Action 1960 
Final Action 1980 

(Applicable to urea only. Do not use for mixed fertilizers.) 

4. Reagents 

(a) Alkaline tartrate soln. — Dissolve 40 g NaOH in 500 mL 
H 2 0, cool, add 50 g NaKC 4 H 4 6 .4f4 2 0, and dil. to 1 L. Let 
stand 1 day before use. 

(b) Copper sulfate soln. — Dissolve 15 g CuS0 4 .5H 2 in 
CCVfree H 2 and dil. to 1 L. 

(c) Biuret. — To recrystallize, weigh ca 10 g reagent grade 
biuret, transfer to 2 L beaker, add 1 L absolute alcohol, and 
dissolve. Cone, by gentle heating to ca 250 mL. Cool at 5° 
and filter thru fritted glass funnel. Repeat crystn and dry final 
product 1 hr at 105-1 10° in oven. Remove from oven, place 
in desiccator, and cool to room temp. 

(d) Biuret std soln. — 1 mg/mL. Dissolve 1 .0000 g recrystd 
biuret in C0 2 -free H 2 and dil. to I L. 



AOAC Official Methods of Analysis (1990) 



Potassium 



23 



B. Preparation of Standard Curve 

Transfer series of aliquots, 2-50 mL, of std biuret soln to 
100 mL vol. flasks. Adjust vol. to ca 50 mL with C0 2 -free 
H 2 0, add 1 drop Me red, and neutze with 0. IN H 2 S0 4 to pink 
color. Add, with swirling, 20 mL alk. tartrate soln and then 
20 mL CuS0 4 soln. Dil. to vol., shake 10 sec, and place in 
H 2 bath 15 min at 30 ±5°. Also prep, reagent blank. Det. A 
of each soln against blank at 555 nm (instrument with 500- 
570 nm filter is also satisfactory) with 2-4 cm cell. Plot std 
curve. 

C. Determination 

Continuously stir <10 g sample contg 30-125 mg biuret in 
150 mL ca 50° H 2 30 min. Filter and wash into 250 mL vol. 
flask, and dil. to vol. Transfer 50 mL aliquot to 100 mL vol. 
flask and proceed as in 960. 04B. 

Refs.: JAOAC 43, 499(1960); 57, 1360(1974); 59, 22(1976); 
60, 323(1977); 62, 153, 330(1979); 63, 222(1980). 

CAS-108-19-0 (biuret) 



976.01 Biuret in Fertilizers 

Atomic Absorption Spectrophotometric Method 

First Action 1976 
Final Action 1980 

A. Apparatus and Reagents 

(a) Atomic absorption spectrophotometer . — IL Model 353 
(Instrumentation Laboratory, Inc., 113 Hartwell Ave, Lexing- 
ton, MA 02173), or equiv., with Cu hollow cathode lamp. 

(b) Copper sulfate soln. — Dissolve 15 g CuS0 4 .5H 2 in 
H,0 and dil. to 1 L. 

(c) Buffer soln.— pH 13.4. Dissolve 24.6 g KOH and 30 g 
KC1 in H 2 and dil. to 1 L. 

(d) Starch soln. — Treat 1 g sol. starch with 10 mL cold 
H 2 0, triturate to thin paste, and pour gradually into 150 mL 
boiling FLO contg 1 g oxalic acid. Boil until soln clears, cool, 
and dil. to 200 mL. Prep, fresh weekly. 

(e) Bromocresol purple indicator. — Dissolve 0.1 g bromo- 
cresol purple in 19 mL 0AN NaOH and dil. to 250 mL with 
H 2 0. 

(f) Biuret.— See 960.04A(c). 

(g) Biuret std soln. — .4 mg/mL. Dissolve 0.4000 g re- 
cry std biuret in warm H 2 0, cool, transfer to 1 L flask, and 
dil. to vol. 

(h) Copper std solns. — Dil. aliquots of Cu stock soln, 
965.09B(b), with H 2 to obtain >4 std solns within range of 
detn, 1-4 |xg Cu/mL final soln. 

B. Preparation of Standard Curve 

Transfer aliquots of biuret std soln contg 0, 2, 4, 6, 8, 10, 
and 12 mg biuret to sep. 100 mL vol. flasks, dil. to ca 30 mL 
with H 2 0, and add 25 mL alcohol to each. While stirring with 
mag. stirrer, add 2 mL starch soln, 10 mL CuS0 4 soln, and 
20 mL buffer soln. Remove stirring bar, rinse, dil. to vol., 
mix thoroly, and let stand 10 min. With vac, filter ca 50 mL 
thru dry 150 mL medium porosity fritted glass funnel into dry 
flask. Transfer 25 mL aliquots of each filtrate to 250 mL vol. 
flasks, acidify with 5 mL IN HCI, and dil. to vol. with H z O. 
Proceed as in 965.09, using std solns, 976.01 A(h), to det. 
complexed Cu in soln by AA spectrophotometry after adding 
equiv. amts of alcohol, KOH soln, buffer soln, and \N HCI. 
Take >3 readings of each soln. From mean value of Cu concn, 
prepare std curve relating mg Cu found to mg biuret added. 
Redet. daily. 



C. Determination 

(a) In urea. — Accurately weigh sample contg <10 mg biu- 
ret, dissolve in H 2 0, transfer to 100 mL vol. flask, add 25 mL 
alcohol, and proceed as in 976. 01B, beginning "While stirring 
with mag. stirrer, ..." From Cu found, calc, biuret concn, 
using std curve. 

(b) In mixed fertilizers . — Transfer accurately weighed sam- 
ple contg <40 mg biuret to 250 mL beaker and add 1 mL H 2 
for each g of sample (5 g max.). Warm, add 65 mL alcohol 
and 7 drops bromocresol purple, and adjust pH to first blue 
color (pH 6-7) with 20% KOH. Place on hot plate, heat to 
bp, cool, and, if pH has changed, make final adjustment to 
first blue. Vac. -filter thru alcohol-washed paper pulp pad into 
100 mL vol. flask. (If filtrate is not clear, improper pH ad- 
justment has been made. Add HCI and readjust to pH 6-7.) 
Wash pad and ppt with alcohol and dil. to vol. with alcohol. 
Transfer 25 mL aliquot to 100 mL vol. flask, and proceed as 
in 976. 01B, beginning "While stirring with mag stirrer, ..." 
From Cu found, calc. biuret concn, using std curve and ap- 
propriate diln factors. (Final aliquot can be varied to give Cu 
concn between 1 and 4 |mg/mL.) 

Refs.: JAOAC 59, 22(1976); 62, 153(1979); 63, 222(1980). 

CAS-108-19-0 (biuret) 



POTASSIUM 

935.02* Potassium in Fertilizers 

Lindo-Gladding Method 

Final Action 
Surplus 1970 



See 2.076-2.078, llthed. 



949.01* Potassium in Fertilizers 

Wet-Digestion Method 

Final Action 
Surplus 1970 

See 2.079-2.080, 11th ed. 



945.02* Recovery of Platinum 

Procedure 

Final Action 
Surplus 1970 

See 2.081-2.083, 11th ed. 



983.02 Potassium in Fertilizers 

Flame Photometric Method 
(Manual or Automated) 

First Action 1983 
Final Action 1985 

(Caution: See safety notes on flame photometer.) 

A. Method Parameters 

Any flame photometer, manual or automated, capable of de- 
tecting K, using Li as internal std, and meeting method per- 
formance characteristics described below, is satisfactory. Sam- 



24 



Fertilizers 



AOAC Official Methods of Analysis (1990) 



pies are extd with ammonium oxalate soln or ammonium citrate 
soln. Appropriate dilns of ext are mixed with LiN0 3 internal 
std soln and aspirated or pumped into flame photometer. La 2 3 
is added to LiN0 3 soln to eliminate the phosphate effect. Final 
soln to be introduced to flame should have the following com- 
position: (a) concn of K 2 in range such that std curve re- 
sponse is linear over that range, (b) const amt of Li in range 
5 to 40 ppm, (c) selected concn of La <1400 ppm, and (d) 
0.2N HN0 3 . Exact concn of LiN0 3 and La 2 3 are optimized 
for particular instrumentation as described in performance 
specifications below. Ratio of K intensity at 768 nm to Li in- 
tensity at 671 nm is detd, and compared with similar ratios 
from std set of >6 stds, prepd from NBS or primary std KH 2 P0 4 . 
Stds are arranged in ascending order and evenly distributed 
thru chosen range. 

&. Preparation of Sample 

(a) Ammonium oxalate extraction. — Weigh 1 g sample into 
500 mL vol. flask, add 50 mL 4% (NH 4 ) 2 C 2 4 and 125 mL 
H 2 0, boil 30 min, and cool. Dil. to vol. with H 2 0, mix, and 
filter or let stand until clear. 

(b) Ammonium citrate extraction from direct available 
phosphorus extract. — Prep, as in 960. 03B. (If solns must be 
held overnight, add 3-4 drops of CHC1 3 .) 

C. Performance Specifications 

System performance criteria. — Detailed example of specific 
instrumental system capable of meeting specified performance 
criteria follows this performance section. It is necessary to ver- 
ify that this or any other particular system meets all of the 
following performance criteria before samples are analyzed. 
Levels specified are to be considered min. acceptable levels. 
Various criteria are written for automated instrument, but should 
also apply to manual instrument systems. 

(a) LiN0 3 concentration level. — Amt of LiN0 3 in final soln 
aspirated into flame is adjusted partly for convenience of in- 
strument parameters, but should be such that Li and K chan- 
nels give roughly equal responses. This can be detd either by 
displaying each channel's output sep., or by displaying ratio 
of K to Li response and then interchanging Li and K filters 
and displaying ratio again. 

Using either procedure, sample midrange K + stds under 
analysis conditions while varying concn of Li + until acceptable 
concn of Li + is found. 

(b) Noise. — Adjust detector output to 90% full scale with 
high std sampled continuously. Noise must be <2% full scale 
peak to peak. Note that some instruments, flow injection anal- 
ysis systems, for example, are not designed to pump samples 
continuously. In this case, substitute repeated sampling for 
continuous sampling, and consider noise to be difference be- 
tween adjacent peak maxima. (Optimum performance on ex- 
ample instrument system described in this method is ca 1 / 2 % 
peak to peak. To reduce noise on example system, stabilize 
flame, stabilize pumping rate, stabilize back-pressure, change 
pump tubes, clean manifold, and /or rework manifold to en- 
sure adequate mixing. To det. min. noise limit of instrument, 
collect system waste soln, connect short length of tubing di- 
rectly to photometer aspirator, and aspirate waste soln directly 
into flame.) 

(c) Carryover. — Adjust detector output to give ca 10% and 
90% full scale response for low and high stds, resp. Sample 
3 high stds followed by 5 low stds on system under analysis 
conditions. Carryover, defined as difference between first low 
std and mean of other low stds, may not be >1% full scale. 
(Optimum performance on example system is negligible car- 
ryover. To reduce carryover on example system, clean man- 
ifold and aspirator, check manifold connections for dead space, 



redesign manifold shortening hydraulic system wherever pos- 
sible, decrease sampling rate, and/or reduce std range.) 

(d) Drift. —Adjust instrument to give detector response ca 
50% full scale with middle std sampled continuously. Sample 
middle std continuously for the time it would take to analyze 
30 samples. For instruments not designed to sample continu- 
ously, draw smooth line thru 30 middle std peaks. Drift may 
not exceed 1% full scale per any 10 sample segment. (Opti- 
mum performance on example system is zero drift. To reduce 
drift on example system, stabilize room and soln temps, adjust 
manifold to maintain const back-pressure, and /or stabilize 
flame.) As long as drift does not exceed 1% per 10 peak level, 
routine data may be further improved by inserting a middle std 
periodically between groups of samples. This allows mathe- 
matical peak ht correction, assuming linear drift. 

(e) Precision.— -With instrument calibrated for 10% and 90% 
full scale for low and high stds, resp., sample 30 middle stds 
under analysis conditions. Range of instrument response may 
not vary >2% full scale. (Optimum performance on example 
system is 0.7% full scale. To improve precision on example 
system, reduce noise, check sampler timing, and/or decrease 
sampling rate.) 

(f) Std curve. — Std curve consists of >6 different stds, evenly 
distributed thru std concn range. Prep, solns from NIST or 
primary std KH 2 P0 4 , dried 2 h at 105°. Include factor for ac- 
tual purity of std material in calcns of std concn. 

With instrument calibrated for ca 10% and 90% response 
for low and high stds, resp., run stds in order of ascending 
concn under analysis conditions. Response should be linear. 
Mathematically perform first degree least squares fit to std curve 
data. Alternatively, use calculator capable of least squares fits. 
First order least squares fit may be performed as follows: As- 
sume that points to be fitted are {X h Y } , (X 2 , Y 2 ), . . . (X n , 
Y„). Calc. means by: 



X = -2Xj 
n 



1 



-2/i 



Slope of least square fitted line is given by: 



bi 



S(Xj - X)(Yi - Y) ax/d - nXY 



(XX^-lMSXj) 2 



%(X } - xf 
Intercept for line is given by: 

b = Y - bjl 
Equation of resulting line is: 

Y = b + bjX 

Using derived equation and individual std responses, calc. 
concn for each std. Compare calcd and known concns for each 
std. Calcd value may not differ from known value by >±2% 
in any one instance. Also, av. of absolute values of those % 
differences may not be >1%. (Optimum performance on ex- 
ample system is 0.75% and 0.37%, resp. To improve std curve 
fit, optimize parameters (b) thru (e) above and /or reduce std 
range.) 

(g) Phosphate effect. — For example system, amt of La 2 3 
in LiN0 3 reagent is sufficient to eliminate phosphate effect 
(depression of instrument response to K by phosphate ion). If 
other than example automated system is used, elimination of 
phosphate effect must be verified. Using KN0 3 , prep. 200 mL 
soln of K 2 with concn equal to twice that of highest std. Pipet 
50 mL of that soln into each of two 100 mL vol. flasks. Dil. 
one to vol. and mix. Add sufficient NH 4 H 2 P0 4 soln to the 
other flask such that concn of P 2 5 will be as high as highest 
concn of P 2 5 anticipated in any sample ext. Dil. to vol. and 
mix. Sample 10 portions of each soln, alternating, under anal- 



AOAC Official Methods of Analysis (1990) 



Potassium 



25 



ysis conditions. Average 10 responses for each soln. Av. re- 
sponses of the 2 solns must not differ from each other by >1%. 
Select min. amt of La 2 3 which will eliminate phosphate ef- 
fect. (Optimum performance of example system is <0.5%. To 
improve performance, adjust amount of La 2 3 .) 

(h) Overall performance of system. — Performance charac- 
teristics mentioned above are worst case examples. A system 
functioning marginally in many categories would probably fail 
the following overall performance check. 

Verify overall performance as follows: Ext and analyze once 
each 20 different Magruder samples, or other similar perfor- 
mance check samples previously detd by interlaboratory study. 
Also ext and analyze 5 independent 1 g portions of NIST or 
primary std KH 2 P0 4 . Randomize Magruder and KH 2 P0 4 sam- 
ple order. Calc. % K 2 0. Av. bias of Magruder results, 2 (Ma- 
gruder grand av. - calcd % K 2 O)/20, must be <±0.1. Av. 
of absolute va]ue of differences must be <0.4. (Optimum val- 
ues on example system are ca ±0.02 and —0.2, resp.) 

For 5 analyses of KH 2 P0 4 , difference between mean of calcd 
% K 2 and known % K 2 must not be >±0.2, and std de- 
viation must not be >0.25. (Optimum values for example sys- 
tem are ±0.1 and 0.15, resp.) 

(i) Ongoing performance checks. — (/) Conduct daily per- 
formance check by analyzing same performance check sample 
at least once in every 60 regular samples, and at least once in 
each run. (2) Repeat (h) above at least twice per year, and 
whenever system has not been used for prolonged periods. 

Example Automated instrument System 

D. Apparatus 

Automatic analyzer. — Auto Analyzer with following mod- 
ules (available from Technicon Instruments Corp.): sampler II 
or IV, pump III, flame photometer IV, and recorder. Com- 
puter or calculator capable of least square fits is desirable. 

E. Reagents 

(a) Ammonium oxalate soln. — Dissolve 40 g (NH 4 ) 2 C 2 4 in 
1 L H 2 0. 

(b) Ammonium citrate soln. — Should have sp. gr. of 1.09 
at 20° and pH of 7.0 as detd potentiometrically. 

Dissolve 370 g cryst. citric acid in 1.5 L H 2 and nearly 
neutze by adding 345 mL NH 4 OH (28-29% NH 3 ). If concn 
of NH 3 is <28%, add correspondingly larger vol. and dissolve 



citric acid in correspondingly smaller vol. H 2 0. Cool, and check 
pH. Adjust with NH 4 OH (1+7) or citric acid soln to pH 7. 
Dil. soln, if necessary, to sp. gr. of 1.09 at 20°. (Vol. will be 
ca 2 L.) Keep in tightly stoppered bottles and check pH from 
time to time. If pH has changed from 7.0, readjust. 

(c) Lithium nitrate soln. — Dissolve 1 .642 g La 2 3 in 30 mL 
HN0 3 , add 0.9935 g dried (2 h at 105°) LiN0 3 and 1 mL 
Flaminox 1% soln (Fisher Scientific Co.), and dil. to 1 L with 
H 2 0. 

(d) Sampler wash and dilution water soln. — Dil. 1 mL 
Flaminox 1% soln to 1 L with H 2 0. 

(e) Potassium std solns.— (1) Stock std soln. — \ mg K 2 0/ 
mL. Dissolve 2.889 g dried (2 h at 105°) KH 2 P0 4 (NIST SRM 
200) in H 2 0, and dil. to 1 L. (2) Working std solns. — 10, 20, 
30, 40, 50, and 55 [ig K 2 0/mL. Accurately measure by buret 
10, 20, and 30 mL stock std soln into 1 L vol. flasks, and 20, 
25, and 27.5 mL into 500 mL vol. flasks. Add 0.2 g (NH 4 ) 2 C 2 4 
per 500 mL final vol. if samples are prepd by ammonium ox- 
alate extn, or add 12 mL ammonium citrate soln per 500 mL 
final vol, if samples are prepd by ammonium citrate extn. Dil. 
to vol. with H 2 and mix. (Add 3 mL CHC1 3 to preserve ci- 
trate std solns for long periods.) 

F. Analytical System 

Assemble manifold as in Fig. 983.02. Use 1.6-2.0 mm id 
glass transmission tubing for all reagent flow upstream from 
Dl fitting. Use clear std pump tubes for air and soln stream 
flow. 

Air and H 2 are combined thru injection fitting (116-0492- 
01). Hard thin-wall polyethylene tubing (ca 0.30 in. id) con- 
nects air bar tubing to injection fitting. Sample is introduced 
immediately downstream thru second injection fitting (194- 
G0 12-01), designed to eliminate double peaks in recorder out- 
put. Mixing of sample and H 2 occurs in double 10-turn coil 
with insert (157-B089). LiN0 3 reagent is introduced thru in- 
sert. Another 10-turn coil (157-0251) further mixes solns. 

Portion of soln is aspirated to flame photometer thru A4 fit- 
ting (116-0200-04). Hard, thin-wall polyethylene tubing (ca 
0.045 in. id) connected to photometer is inserted and glued to 
tee arm of A4 fitting. Remaining unaspirated soln is drawn 
thru double 10-turn mixing coil (157-9248-01) and thru Dl 
fitting (116-0203-01). Large diam. branch of Dl fitting leads 
to pump and waste. Small diam. branch of Dl fitting is con- 







0.23 ML /MIN 


SAMPLE 




c 

D 0000 0000 


A 1.00 ML /MIN 


WATER 


f °l 


owo 


B 


0.32 ML/ MIN 


AIR 




0.80 ML/ MIN 


UNO* 


SAMPLER IV 


7 


~ .. 2.00 ML/ MIN 


WATER 


1 V w 

TO WA 
E 0000 


~„ 0.80 ML/MIM 




0000 G 








F 


TO 6' 


OF .030 
















TY60N TUBING 








-<r 










(J) 




1 TT 1 








ywj 








FLAME 
PHOTOMET 


ER 




RECORDER 







FIG. 983.02— Manifold for K 2 in fertilizers. A, injection fitting 116-0492-01; B, injection fitting 194-G012-01; C, double 10-turn 
coil with insert 157-B089; D, 10-turn coil 157-0251; E, A4 fitting 116-0200-04; F, double 10-turn coil 157-0248-01; G, D1 fitting 

116-0203-01 



NORTHFAST WISCONSIN TFFhNsCAL C0U.E(it 

LEARNING RESOURCE CENTER 

GBEEM BAY, Wl 54307 



26 



Fertilizers 



AOAC Official Methods of Analysis (1990) 



nected to 6 ft (I . .83 m) of Tygon tubing (0.030 in. id) to waste. 
Dl. fitting is oriented with small diam. branch low, so that only 
soln, and no air, enters 0.030 in. tubing. This establishes const 
back-pressure and therefore stable aspiration conditions at flame 
photometer. 

G. Startup and Shutdown Procedures 

Start system and place reagent lines in proper solns. Let 
equilibrate 30 min before beginning calibration. Adjust flame 
photometer as follows: (/) damping control to damp 3 posi- 
tion; (2) flame ht of main cone ca 4 cm; (3) atomizer adjust 
control set to give atomization rate of ca 1.3-1.4 mL/min. 
Rate of atomization is detd by subtracting rate of flow to waste 
from rate of flow upstream from (disconnected) A4 fitting. Use 
0.3 and 0.6 neut. density filters for Li and K detectors, resp. 

Initially it may be necessary to manually fill system down- 
stream from A4 fitting with H 2 0, making certain that 6 ft of 
0.030 tubing is filled. System is shut down after pumping H 2 
thru reagent lines 5:15 min. 

H. Checkout and Calibration 

After equilibration, pump 10 (xg K 2 0/mL std thru system 
and adjust baseline control of photometer to read 10% full scale. 
Pump 55 jmg K 2 0/mL std thru system and adjust std calibra- 
tion control to read 90% full scale. If noisy conditions exist, 
check for aspiration of air at A4 fitting, or check for air en- 
tering lower arm of Dl fitting. If drift exists, check room and 
solns for temp, stability. Std curves should be virtually linear. 

/. Determination 

Pipet aliquots of sample solns. Table 983.02, into 250 mL 
vol. flask, dil. to vol. with H 2 0, and mix 15 times. For 10 
mL aliquots of citrate extns, add 4 mL ammonium citrate so In 
to aliquots before dilg to vol. Run samples in groups of 10. 
Place 10 thru 55 fxg K 2 0/mL stds in order in sampler, pre- 
ceded by extra 10 (xg/mL std. Place 30 |JLg/mL std after every 
10th sample, to be used for drift correction. End series with 
two 30 |xg/mL stds. Sample at rate of 40/h, 2:1 sample-to- 
wash ratio. 

J. Calculations 

Correct sample peak hts for drift. Correct peak hts of first 
10 samples as follows: 

// c = // - |(O t - Z> )/14][L + 3] 

where H c — corrected peak ht; H = uncorrected peak ht; 
£>! - ht of first drift correction std; D = ht of 30 \xg std in 
initial std sequence; and L - position No. of sample peak to 
be corrected. Correct subsequent sample peak hts as follows: 

# c = H - [D x - D ] - \{D y - D X )/11][P] 

where D x = ht of drift std preceding sample to be corrected; 
D y = ht of drift std following sample to be corrected; and 
P = position No. of sample within group of 10. 

Calc. least squares fitted curve of emission against K 2 concn. 
Calc. (xg K 2 0/mL of corrected peak hts from equation: 

% K 2 = (|xg K 2 0/mL X 12.5) /(aliquot X g sample) 

Ref.: JAOAC 66, 1242(1983). 



Table 983.02 K 2 Aliquots 




K 2 Expected, % 


Aliquot, mL 


<2 

2-6.49 
6.50-19.99 
>20 


250 (no diln) 
100 

30 

10 



CAS-7440-09-7 (potassium) 
CAS- 12 136-45-7 (potassium oxide) 



955.06* Potassium in Fertilizers 

Flame Photometric Method 
Final Action 
Surplus 1986 

See 2.108-2.113, 14th ed. 



971 .01 • Potassium in Fertilizers 

Automated Flame Photometric Method 

First Action 1971 

Final Action 1973 

Surplus 1986 

See 2.114-2.118, 14th ed. 



958.02 Potassium in Fertilizers 

Volumetric Sodium Tetraphenylboron Method I 

First Action 1958 
Final Action 1960 

(Caution: See safety notes on formaldehyde.) 

A. Reagents 

(a) Formaldehyde soln. —31%. 

(b) Sodium hydroxide soln. — 20%. Dissolve 20 g NaOH in 
100 mL H 2 0. 

(c) Sodium tetraphenylboron (STPB) soln.— Approx. 1.2%. 
Dissolve 12 g NaB(C 6 H 5 ) 4 in ca 800 mL H 2 0. Add 20-25 g 
Al(OH) 3 , stir 5 min, and filter (Whatman No. 42 paper, or 
equiv.) into 1 L vol. flask. Rinse beaker sparingly with H 2 
and add to filter. Collect entire filtrate, add 2 mL 20% NaOH, 
dil. to vol. with H 2 0, and mix. Let stand 48 hr and stdze. 
Adjust so that 1 mL STPB = 1% K 2 0. Store at room temp. 

(d) Benzalkonium chloride (BAC) soln, — Approx. 0.625%. 
Dil. 38 mL 17% Zephiran chloride (Winthrop Laboratories; 
also available at local pharmacies as benzalkonium chloride) 
to 1 L with H 2 0, mix, and stdze. Cetyltri methyl ammonium 
bromide may be substituted for Zephiran chloride. If other concn 
is used, adjust vol. 

(e) Clayton Yellow (Titan Yellow; Colour Index No. 
19540).— 0.04%. Dissolve 40 mg in 100 mL H 2 0. 

B. Standardization of Solutions 

(a) BAC soln.— To 1.00 mL STPB soln in 125 mL erlen- 
meyer, add 20-25 mL H 2 0, 1 mL 20% NaOH, 2.5 mL HCHO, 
1.5 mL 4% (NH 4 ) 2 C 2 0^ and 6-8 drops indicator, (e). Titr. to 
pink end point with BAC soln, using 10 mL semimicro buret. 
Adjust BAC soln so that 2.00 mL - 1.00 mL STPB soln. 

(b) Sodium tetraphenylboron soln. — Dissolve 2.500 g 
KH 2 P0 4 in H 2 in 250 mL vol. flask, add 50 mL 4% 
(NH 4 ) 2 C 2 4 soln, dil. to vol. with H 2 0, and mix. (It is not 
necessary to bring to boil.) Transfer 15 mL aliquot (51.92 mg 
K 2 0, 43.10 mg K) to 100 mL vol. flask; add 2 mL 20% NaOH, 
5 mL HCHO, and 43 mL STPB reagent. Dil. to vol. with 
H 2 0, mix thoroly, let stand 5—10 min, and pass thru dry filter. 
Transfer 50 mL aliquot of filtrate to 125 mL erlenmeyer, add 
6-8 drops indicator, (e), and titr. excess reagent with BAC 
soln. Calc. titer as follows: 



AOAC Official Methods of Analysis (1990) 



Other Elements 27 



F = 34.61/(43 mL - mL BAC) - % K 2 0/mL STPB reagent 

Factor F applies to all fertilizers if 2.5 g sample is dild to 250 
mL and 15 mL aliquot is taken for analysis. If results are to 
be expressed as K rather than as K 2 0, substitute 28.73 for 
34.61 in calcg F. 

C. Determination 

Place 2.5 g sample (1.25 g if K 2 >50%) in 250 mL vol. 
flask, add 50 mL 4% (NH 4 ) 2 C 2 4 and 125 mL H 2 0, and boil 
30 min. (If org. matter is present, add 2 g K-free C before 
boiling.) Cool, dil. to vol. with H 2 0, mix, and pass thru dry 
filter or let stand until clear. Transfer 15 mL aliquot sample 
soln to 100 mL vol. flask and add 2 mL 20% NaOH and 5 
mL HCHO. Add 1 mL std STPB soln for each 1% K 2 ex- 
pected in sample plus addnl 8 mL excess to ensure complete 
pptn. Dil. to vol. with H 2 0, mix thoroly, let stand 5-10 min, 
and filter thru dry paper (Whatman No. 12 or equiv.). Transfer 
50 mL filtrate to 125 mL erlenmeyer, add 6-8 drops indicator, 
(e), and titr. excess reagent with std BAC soln. 

% K 2 in sample = (mL STPB added - mL BAC) X F 

where F = % K 2 0/mL STPB reagent. (Multiply by 2 if 1.25 
g sample was used.) 

Refs: Anal. Chem. 29, 1044(1957); 30, 1882(1958). JAOAC 
41, 533(1958); 43, 472(1960). 

CAS-7440-09-7 (potassium) 
CAS- 13547- 17-6 (potassium oxide) 



D. Determination 

Transfer 25 mL aliquot of sample soln to 100 mL vol. flask. 
(If org. matter is present, treat 100 mL portion with 1 g K- 
free C and filter before transferring aliquot.) Add 8 mL HCHO 
first and then 5 mL 20% NaOH soln, and wash down sides of 
flask with H 2 0. Swirl and add 1 mL STPB for each 1.5 mg 
K 2 expected in sample aliquot plus addnl 8 mL excess to 
ensure complete pptn. Dil. to vol. with H 2 0, mix thoroly, let 
stand 5-10 min, and pass thru dry filter (Whatman No. 12, 
or equiv.). Transfer 50 mL aliquot filtrate to 125 mL erlen- 
meyer, add 6-8 drops indicator, 958*02A(e), and titr. excess 
reagent with std BAC soln. 

% K 2 in sample - (mL STPB added - mL BAC) x F x 2 

Calcn applies to all fertilizers if 1 g sample is dild to 500 
mL and 25 mL aliquot is taken for analysis. 

Ref: JAOAC 52, 566(1969). 

CAS-7440-09-7 (potassium) 
CAS- 13547- 17-6 (potassium oxide) 



OTHER ELEMENTS 

965.09 Nutrients (Minor) in Fertilizers 

Atomic Absorption Spectrophotometric Method 

First Action 1965 
Final Action 1969 



969.04 Potassium in Fertilizers 

Volumetric Sodium Tetraphenylboron Method II 

First Action 1969 
Final Action 1970 

(For use with sample prepd for available P detn) 
(Caution: See safety notes on formaldehyde.) 

A Reagents 

See 958.02A(a), (b), (c), (d), and (e). 

B. Standardization of Solutions 

(a) BAC soln.— in 125 mL erlenmeyer, add 2.5 mL neut. 
NH 4 citrate soln, 963.03A(a), 15-20 mL H 2 0, 4 mL HCHO, 
and 2.5 mL 20% NaOH soln. Swirl; then add 4.00 mL STPB 
soln and 6-8 drops indicator, 958.02A(e). Titr. to pink end 
point with BAC soln, using 10 mL semimicro buret. Adjust 
BAC soln so that 2.00 mL - 1.00 mL STPB soln. 

(b) Sodium tetraphenylboron soln. — Dissolve 1.4447 g pri- 
mary std KH 2 P0 4 in H 2 in 500 mL vol. flask, add 100 mL 
neut. NH 4 citrate soln, 963.03A(a), dil. to vol. with H 2 0, and 
mix. Transfer 25 mL aliquot (25.00 mg K 2 0, 20.75 mg K) to 
100 mL vol. flask; add 8 mL HCHO and 5 mL 20% NaOH, 
swirl, and add 25 mL STPB reagent. Dil. to vol. with H 2 0, 
mix thoroly, let stand 5-10 min, and pass thru dry filter. Transfer 
50 mL aliquot of filtrate to 125 mL erlenmeyer, add 6-8 drops 
indicator, 958.02A(e), and titr. excess reagent with BAC soln. 
Calc. titer as follows: 

F = 25 mg K 2 0/(25 mL STPB - mL BAC) 

= mg K 2 0/mL STPB reagent 

if results are to be expressed as K rather than K 2 0, substitute 
20.75 for 25 in calcg F. 

C. Preparation of Sample 
Prep, as in 960.03B. 



(Caution: See safety notes on atomic absorption spectropho- 
tometer.) 

A. Apparatus and Reagent 

(a) Atomic absorption spectrophotometer. — Several com. 
models are available. Since each design is somewhat different, 
with varying requirements of light source, burner flow rate, 
and detector sensitivity, only general outline of operating pa- 
rameters is given in Table 965.09. Operator must become fa- 
miliar with settings and procedures adapted to his own app. 
and use table only as guide to concn ranges and flame con- 
ditions. 

(b) Disodium EDTA soln.— 2.5%. Dissolve 25 g Na 2 H 2 
EDTA in 1 L H 2 and adjust to pH 7.0 with 5N NaOH, using 
pH meter. 

B. Standard Solutions 

(Do not use <2 mL pipets or <25 mL vol. flasks. Automatic 

diln app. may be used. Prep, std solns in 0-20 \xg range fresh 

daily.) 

(a) Calcium solns. — (J) Stock soln. — 25 |xg Ca/mL. Dis- 
solve 1.249 g CaC0 3 in min. amt 3/V HC1. Dil. to 1 L. Dil. 
50 mL to 1 L. (2) Working std solns.— 0, 5, 10, 1.5, and 20 
|xg Ca/mL contg 1% La. To 25 mL vol. flasks add 0, 5, 10, 
15, and 20 mL Ca stock soln. Add 5 mL La stock soln and 
dil. to 25 mL. 

(b) Copper stock soln. — 1000 juig Cu/mL. Dissolve 1.000 
g pure Cu metal in min. amt HN0 3 and add 5 mL HO. Evap. 
almost to dryness and dil. to 1 L with 0. 1W HC1. 

(c) Iron stock soln. — 1000 |xg Fe/mL. Dissolve 1.000 g pure 
Fe wire in ca 30 mL 6N HC1 with boiling. DiJ. to 1 L. 

(d) Lanthanum stock soln. — 50 g La/L. Dissolve 58.65 g 
La 2 3 in 250 mL HC1, adding acid slowly. Dil. to 1 L. 

(e) Magnesium stock soln. — 1000 |xg Mg/mL. Place 1.000 
g pure Mg metal in 50 mL H 2 and slowly add 10 mL HCL 
Dil. to 1 L. 



28 



Fertilizers 



AOAC Official Methods of Analysis (1990) 



(f ) Manganese stock soln. — 1000 |xg Mn/mL. Dissolve L582 
g Mn0 2 in ca 30 mL 67V HO. Boil to remove CI and dil. to 
1 L. 

(g) Zinc stock soln, — 1000 {xg Zn/mL. Dissolve 1.000 g 
pure Zn metal in ca 10 mL 67V HCL Dil. to 1 L. 

(h) Other std solns. — Dil. aliquots of solns (b), (c), (e), 
(f ), and (g) with 0.57V HC1 to make >4 std solns of each ele- 
ment within range of detn. 

C. Preparation of Sample Solutions 

{Caution: See safety notes on wet oxidation, hydrofluoric acid, 
and perchloric acid.) 

(a) Inorganic materials and mixed fertilizers. — Dissolve 1 .00 
g well ground sample in 10 mL HC1 in 150 mL beaker. Boil 
and evap. soln nearly to dryness on hot plate. Do not bake 
residue. Redissolve residue in 20 mL 27V HC1, boiling gently 
if necessary. Filter thru fast paper into 100 mL vol. flask, 
washing paper and residue thoroly with H 2 0. Measure ab- 
sorption of soln directly, or dil. with 0.57V HC1 to obtain solns 
within ranges of instrument. If Ca is to be detd, add enough 
La stock soln to make final diln 1% La (i.e., 5 mL La to 25 
mL flask, 20 mL to 100 mL flask, etc.). 

(b) Fertilizers containing organic matter ( tankage y corn- 
cobs, cottonseed hulls, etc.). — Place 1 .00 g sample in 150 mL 
beaker (Pyrex, or equiv.). Char on hot plate and ignite 1 hr 
at 500° with muffle door propped open to allow free access of 
air. Break up cake with stirring rod and dissolve in 10 mL 
HC1 as in (a). 

(c) Fertilizers containing fritted trace elements. — Dissolve 
<1.00 g well ground sample in 5 mL HC10 4 and 5 mL HF. 
Boil and evap. to dense HC10 4 fumes. Dil. carefully with H 2 0, 
filter, and proceed as in (a). Alternatively, dissolve sample in 
10 mL HCI, 5 mL HF, and 10 mL MeOH. Evap. to dryness. 
Add 5 mL HC1 and evap. Repeat HCI addn and evapn. Dis- 
solve residue as in (a). (Normally Pt ware should be used; 
Pyrex or other glassware may be used if Na, K, Ca, and Fe 
are not to be detd.) 

(d) For manganese . — (J ) Acid-soluble, for both Mn + ~ and 
Mn +4 .—See (a), (b), and (c), and 972.02(b). (2) Acid-soluble, 
for Mn' 2 only.— See 972.02(a), 940.02, and 941.02*. (3) 

Water-soluble, for Mn +2 only. — See 972.03. 

(e) For iron and zinc. — (7) Aqueous extraction. — Place 1.00 
g sample in 250 mL beaker, add 75 mL H 2 0, and boil 30 min. 
Filter into 100 mL vol. flask, washing paper with H 2 0. Dil. 
to vol. and redil. if necessary. (2) Chelation extraction. — Place 
1.00 g sample in 250 mL beaker, and add 5 cm (2") mag. 
stirrer bar and 100 mL 2.5% EDTA soln. Stir exactly 5 min, 
and filter thru Whatman No. 41 paper, or equiv. If filtrate is 
cloudy, refilter immediately thru fine paper (Whatman No. 5, 
or equiv.). Redil., if necessary, with 0.57V HCI. 

D. Determination 

(P interferes in Ca and may interfere in Mg detn with air-C 2 H 2 

burners. Eliminate interference by adding La stock soln to std 

and sample solns so that final dilns contain 1% La. P does not 

interfere with Ca detn when N 2 0-C 2 H 2 flame is used.) 

Set up instrument as in Table 965.09, or previously estab- 
lished optimum settings for app. to be used. Less sensitive 
secondary lines (Gatehouse, and Willis, Spectrochim. Acta 17, 
710(1961)) may be used to reduce necessary diln, if desired. 
Read ^4 std solns within anal, range before and after each 
group of 6-12 samples. Flush burner with H 2 between sam- 
ples, and re-establish absorption point each time. Prep, cal- 
ibration curve from av. of each std before and after sample 
group. Read concn of samples from plot of absorption against 
fxg/mL. 



Table 965.09 Operating Parameters 





Wave- 




Range, 




Element 


length, A 


Flame 


^g/mL 


Remarks 


Ca 


4227 


Rich Air-C 2 H 2 


2-20 


1% La, 1% HCI 




4227 


Rich N 2 OC 2 H 2 


2-20 


Requires special 
burner 


Cu 


3247 


Air-C 2 H 2 


2-20 




Fe 


2483 


Rich Air-C 2 H 2 


2-20 




Mg 


2852 


Rich Air-C 2 H 2 


0.2-2 


May need La 


Mn 


2795 


Air-C 2 H 2 


2-20 




Zn 


2138 


Air-C 2 H 2 


0.5-5 





E. Calculations 

% Element = (fxg/mL) x (F/sample wt) x 10~ 4 
F — mL original diln X ml final diln/mL aliquot, if original 
100 mL vol. is diJd. 

Refs.: JAOAC 48, 406, 1100(1965); 50, 401(1967); 51, 
847(1968); 58, 928(1975). 

CAS-7440-70-2 (calcium) 
CAS-7440-50-8 (copper) 
CAS-7439-89-6 (iron) 
CAS-7439-95-4 (magnesium) 
CAS-7439-96-5 (manganese) 
CAS-7440-66-6 (zinc) 



949.02 Boron (Acid-Soluble) in Fertilizers 
Titrimetric Method 
Final Action 

A. Apparatus 

Use high sensitivity glass electrode pH meter for titrn. Use 
assembly with burets, electrodes, and mech. stirrer, arranged 
for convenient use with 250 mL beaker. Use ordinary 50 mL 
burets for the 0.0257V NaOH and 0.027V HCL 

B. Reagents 

(a) Boric acid std soln. — Dissolve 1 g H 3 B0 3 in H 2 and 
dil. to 1 L. 1 mL = 0.1748 mg B. 

(b) Sodium hydroxide std soln. — C0 2 -free, ca 0.0257V. Stdze 
as follows: Pipet 25 mL std H 3 B0 3 soln into 250 mL beaker, 
add 3.0 g NaCl, acidify to Me red, dil. to 150 mL, boil to 
expel C0 2 , cool, and titr. potentiometrically as in 949. 02C. 
Det. blank by repeating titrn, substituting 25 mL H 2 for H3BO3 
soln. Calc. B equivalence as follows: 

mg B/mL = 4.369/[(mL NaOH soln) - (mL blank)] 

Protect from atm. C0 2 by soda-lime tubes or other suitable 
means. 

(c) Methyl red indicator. — Dissolve 0. 1 g Me red in 50 mL 
alcohol, dil. to 100 mL with H 2 0, and filter if necessary. 

C. Determination 

Weigh sample within 1 mg (1.0 g for up to 0.45% B, smaller 
samples for above that content) and place in 250 mL beaker. 
Add ca 50 mL H 2 and 3 mL HCI. Heat to bp and keep hot 
until carbonates are decomposed. Keep soln hot but do not boil 
during following phosphate removal: 

Add 10% Pb(N0 3 ) 2 soln, usually 10 mL, or 1 mL for each 
1.2% P 2 5 if P 2 5 content is known to be > 1 2% . Add NaHC0 3 , 
little at time, until soln approaches neutrality (often observed 
by formation of white ppt in addn to insol . matter already pres- 
ent). Add few drops Me red and continue adding NaHC0 3 
gradually until just alk. to Me red (yellow or very slightly or- 



AOAC Official Methods of Analysis (1990) 



Other Elements 



29 



ange). Keep mixt. hot but not boiling (H 2 bath or steam bath 
is best) 30 min, adding addnl small amts of NaHC0 3 if needed 
to keep same indicator color. (If indicator is bleached by ni- 
trate present, add more; if color is obscured by org. matter, 
use external spot tests to follow neutzn.) After neutzn and 
heating, 40-50 mL soln should remain. 

Filter hot soln into 250 mL beaker and wash solids thoroly 
with hot H 2 0. Acidify filtrate with few drops HO and boil 
briefly to expel most of C0 2 . Neutze hot soln with 0.5N NaOH, 
and reacidify with 0.5 A" HO, using 0.3-0.5 mL excess. DiJ. 
to ca 150 mL and boil gently few min to expel remaining C0 2 . 
Cool to room temp, in running H 2 0. Roughly neutze mixt., 
using C0 2 -free 0.5N NaOH, and place beaker in titrn assembly 
with electrodes and stirrer immersed. Start stirrer and adjust 
pH to exactly 6.30 by adding 0.0257V NaOH or 0.027V HC1 as 
required. (When properly adjusted, pH should be steady; drift- 
ing usually is due to incomplete removal of C0 2 .) When read- 
ing of pH 6.30 is steady, read 0.025/V NaOH buret, add 20 g 
mannitol or cryst. D-sorbitol, and titr. with 0.0257V NaOH to 
pH 6.30. (Conveniently done with slidewire type instrument 
by opening pH meter circuit when mannitol is added, leaving 
scale setting at 6.30, closing circuit again when indicator color 
shows that end point is being approached, and carefully adding 
std NaOH soln until galvanometer needle returns to zero. 
Somewhat slow approach to equilibrium, characteristic of glass 
electrode, can be anticipated with practice so as not to overrun 
end point.) When end point is reached, read buret again. Ob- 
tain reagent blank by repeating detn with all reagents but with- 
out sample. 

% B = (mL NaOH soln in detn - mL blank) 

x (mg B/mL NaOH soln)/(10 x g sample) 

Refs.: JAOAC 32, 422(1949); 33, 132(1950); 36, 623(1953); 
38, 407(1955). 

CAS-7440-42-8 (boron) 



949.03 Boron (Water-Soluble) in Fertilizers 
Titrimetric Method 
Final Action 

(Not applicable in presence of >5% urea or urea-formaldehyde 

resins) 

Weigh 2.5 g sample into 250 mL beaker. Add 125 mL H 2 0, 
boil gently ca 10 min, and filter hot thru Whatman No. 40 
paper, or equiv., into 400 mL beaker. Wash solids well with 
6 portions hot H 2 and dil. to >200 mL with H 2 0. Heat fil- 
trate just to bp. Add 15 mL 10% BaCl 2 soln to ppt sulfates 
and phosphates, and add powd Ba(OH) 2 , cautiously with stir- 
ring, until just alk. to phthln, avoiding large excess. Boil in 
open beaker >60 min to expel NH 3 . (Samples colored by org. 
matter should be boiled longer.) If necessary, add H 2 to keep 
vol. to >150 mL. Add and stir 1-2 teaspoonfuls Filter-Cel or 
other inert filtering aid, and filter with suction thru packed 
paper pads into 500 mL Pyrex erlenmeyer, Wash ppt 6 times 
with hot boiled H 2 0. (Avoid too large wash vols which in- 
crease vol. in flask to point of dangerous bumping in next step.) 

Make filtrate just colorless to phthln with HO (1 + 5), add 
Me red, and make just pink with the acid. Add 5 or 6 boiling 
stones and stirring rod, cover with watch glass, and boil 5 min 
to remove C0 2 . Cool in cold H 2 while covered. Wash cover 
glass, stirrer, and sides of flask. Titr. to yellow of Me red with 
std 0.05N NaOH, 936.16. Add 20 g D-mannitol and 1 mL or 
more phthln, shake, and wash down sides of flask. Titr. to 
pink end point. Det. blank in exactly same manner as sample. 



1 m'L0.05iVNaOH 

- 0.000540 g B or 0.00477 g Na 2 B 4 7 . 10H 2 O 

Or, (Titer - blank) x factor 

= lb Na 2 B 4 O 7 .10H 2 O/ton(f actor = 3.807 for 0.05/V NaOH). 

Refs.: JAOAC 32, 422(1949); 33, 132(1950); 36, 623(1953); 
38, 407(1955). 

CAS-7440-42-8 (boron) 



982.01 Boron (Acid- and Water-Soluble) 

in Fertilizers 

Spectrophotometric Method 

First Action 1982 
Final Action 1985 

A. Apparatus and Reagents 

(a) Spectrophotometer. — Beckman Model 24/25 (replace- 
ment model DV-64), or equiv. 

(b) Precision pipet. — 100 (jlL Sherwood Lancer (Monoject 
Scientific, 200 Express St, Plainview, NY 11803), or equiv. 

(c) Dispenser pipet. — Automatic (Repipet, Lab industries, 
Inc., 620 Hearst Ave, Berkeley, CA 94710), or equiv., 5 mL 
capacity. 

(d) Boron std solns. — (J) Stock soln. — 100 jxg/mL. Dis- 
solve 0.5716 g boric acid in H 2 and dil. to 1 L with H 2 0. 
Mix well and transfer to plastic bottle. (2) Working solns. — 
0, 5, 10, 15, 20, 25, 30, and 45 |ig/mL. Pipet 0, 5, 10, 15, 
20, 25, 30, and 45 mL stock soln into sep. 100 mL vol. flasks, 
dil. to vol. with 1% HO, mix well, and transfer to plastic 
bottles. Solns are stable. 

(e) Azomethine H color reagent. — Dissolve 0.9 g azome- 
thine H (Pierce Chemical Co.) and 2.0 g ascorbic acid in 100 
mL H 2 0. Store in refrigerator and discard after 14 days. 

(f) Buffer-masking soln. — Dissolve 140 g ammonium ace- 
tate, 10 g potassium acetate, 4 g nitrilotri acetic acid, disodium 
salt 99+% (Aldrich Chemical Co., Inc., No. 10629-1), 10 g 
(ethylenedinitrilo) tetraacetic acid, and 350 mL 10% acetic acid 
(v/v) in H 2 and dil. to 1 L with H 2 0. Soln is stable. 

(g) Color developing reagent. — Place 35 mL azomethine H 
color reagent and 75 mL buffer- masking soln into 250 mL vol. 
flask and dil. to vol. with H 2 0. Prep, fresh daily. 

B. Preparation of Sample Solutions 

(a) Acid-soluble boron. — Weigh 2.00 g sample into 100 mL 
vol. flask, add 30 mL H 2 and 10 mL HO, stopper, and shake 
15 min. Dil. to vol. with H 2 0, mix well, and filter immedi- 
ately into plastic bottle. Dil. as necessary, so final soln for 
color measurement falls within std curve. 

(b) Water-soluble boron. — Weigh 2.00 g sample into 250 
mL beaker, add 50 mL H 2 0, and boil ca 10 min. Filter hot 
thru Whatman No. 40 paper, or equiv., into 100 mL vol. flask. 
Wash ppt 6 times with hot, boiled H 2 until vol. in flask is 
ca 95 mL. Cool, add 1.0 mL HO, dil. to vol. with H 2 0, and 
mix. Transfer to plastic bottle immediately; dil. as necessary 
so final soln for color measurement falls within std curve. 

C. Determination 

Pipet 100 |ulL aliquots of 0, 5, 10, 15, 20, 25, 30, and 45 
jxg/mL std and 100 jjiL aliquots of sample solns into sep. 10 
mL erlenmeyers. Add 5.0 mL color developing reagent by au- 
tomatic pipet dispenser (5 mL pipet is suitable but slower) and 
let stand 1 h at room temp. Transfer to 1 cm cell and read A 
at 420 nm against H 2 0. Correct for reagent blank (0 mg/mL 
std). Construct std curve by plotting A against |xg/mL stds and 
read concns (jig/mL) of sample solns from std curve. 



30 



Fertilizers 



AOAC Official Methods of Analysis (1990) 



D. Calculation 

Boron, % = (|mg/mL from std curve) X diln factor 

x (100/g sample) x 1(T 6 

Ref.: J AOAC 65, 234(1982). 

CAS-7440-42-8 (boron) 

945.03 Calcium (Acid-Soluble) in Fertilizers 
Titrimetric Method I 
Final Action 

(Presence of other analytes pptd by oxalate, such as Ba and 
Sr, will cause pos. bias in results.) 

Weigh 2.5 g sample into 250 mL vol. flask, add 30 mL 
HN0 3 and 10 mL HC1, and boil 30 min. Cool, dil. to vol., 
mix, and filter if necessary. Transfer 25 mL aliquot to beaker 
and dil. to 100 mL. Add 2 drops bromophenol blue (grind 0.1 
g bromophenol blue powder with 1.5 mL 0.1N NaOH and dil. 
to 25 mL with H 2 0). Add NH 4 OH (1+4) until indicator changes 
from yellow to green (not blue). If overrun, bring back with 
HC1 (1 +4). (This gives pH of 3.5-4.0.) Dil. to 150 mL, bring 
to bp, and add 30 mL satd hot (NH4) 2 C 2 04 soln slowly, stirring 
constantly. If color changes from green to blue or yellow again, 
adjust to green with HC1 (1+4). If yellow, adjust with NH 4 OH 
to green. Digest on steam bath 1 hr, or let stand overnight, 
and cool to room temp. Filter supernate thru quant, paper, 
gooch, or fritted glass filter, and wash ppt thoroly with NH 4 OH 
(1+50). Place paper or crucible with ppt in original beaker 
and add mixt. of 125 mL H 2 and 5 mL H 2 S0 4 . Heat to >70° 
and titr. with 0AN KMn0 4 until first slight pink appears. Cor- 
rect for blank and calc. to Ca. 



965.1 0* Calcium (Acid-Soluble) 

in Fertilizers 
Titrimetric Method II 

Final Action 
Surplus 1980 

See 2.142, 14th ed. 

945.04 Calcium (Acid-Soluble) in Fertilizers 
Atomic Absorption Spectrometric Method 
Final Action 

See 965.09. 



955.07* 



Carbon (Carbonate) 
in Fertilizers 

Final Action 
Surplus 1970 



See 2.107-2.108, 11th ed. 



928.02 Chlorine (Water-Soluble) 

in Fertilizers 

Final Action 

A. Reagents 

(a) Silver nitrate std soln. — Dissolve ca 5 g recrystd AgN0 3 
in H 2 and dil. to 1 L. Stdze against pure, dry NaCl and adjust 
so that 1 mL soln = 0.001 g CI. 

(b) Potassium chromate indicator. — See 941.18B(b), 



B. Determination 

Place 2.5 g sample on 11 cm filter paper and wash with 
successive portions boiling H 2 until washings total nearly 250 
mL, collecting filtrate in 250 mL vol. flask. Cool, dil. to vol. 
with H 2 0, and mix well. Pipet 50 mL into 150 mL beaker, 
add 1 mL K 2 Cr0 4 indicator, and titr. with std AgN0 3 soln to 
permanent red of Ag 2 Cr0 4 . 

Refs.: JAOAC 11, 34,201(1928); 16, 69(1933). 

CAS-7782-50-5 (chlorine) 



965.11 Cobalt in Fertilizers 

Cotorimetric Method 

First Action 1965 
Final Action 1969 

{Caution: See safety notes on nitric acid and perchloric acid.) 

A. Reagents 

(Use H 2 free of interfering elements. Check by shaking 2 

drops 0.01% dithizone in CC1 4 with 10 mL H 2 0. CC1 4 phase 

should remain green.) 

(a) Ternary acid mixture. — See 960.03A(b). 

(b) Ammonium hydroxide. — Use fresh stock. (Reagent be- 
comes contaminated with heavy metals on prolonged storage 
in glass.) 

(c) Isoamyl acetate. — Distd. 

(d) 2-Nitroso-l-naphthol soln. —0.05%. Dissolve 0.05 g 2- 
nitroso-1-naphthol in 8 drops 1W NaOH and 1 mL ILO. Add 
50-60 mL H 2 and 6.5-7 mL NH 4 OH, and dil. to "lOO mL 
with H 2 0. Divide into 2 ca equal parts and wash each part 
twice in 100 mL centrf. tube with 20 mL isoamyl acetate. Shake 
30 sec and centrf. after each addn. (It may be necessary to 
remove part of aq . phase to ensure complete removal of for- 
eign matter at interface.) 

(e) Cobalt std so Ins. — (J) Stock soln, — 200 \xg Co/mL. 
Dissolve 0.0808 g CoCl 2 .6H 2 in H 2 and dil. to 100 mL. 
(2) Working soln. — 2 \xg Co/mL. Dil. I mL stock soln to 100 
mL with H 2 0. 

B, Determination 

Slowly add 20 mL ternary acid mixt. to 2.00 g pulverized, 
mixed fertilizer in 150 mL beaker. Cover with watch glass and 
digest on steam bath overnight. Transfer to hot plate and heat 
covered until dense white fumes appear. (At this point HNO3 
will have been expelled. Take care not to lose significant amts 
of H.CIO4.) Dil. sample contg undissolved residue with H 2 0, 
transfer to 50 mL vol. flask, and dil. to vol. Transfer to 100 
mL centrf. tube and centrf. 5 min at 2000 rpm. Transfer ali- 
quot, contg 2-5 fxg Co, to 50 mL g-s centrf. tube. Add 10 
mL 20% diammonium citrate soln and 2 drops phthln. Adjust 
pH carefully to distinct pink with NH 4 OH (1 + 1) and add suc- 
cessively 1 mL 10% Na 2 S 2 3 soln, 2 mL 2-nitroso-l-naphthol 
soln, and 5 mL isoamyl acetate. (Only isoamyl acetate addn 
requires high degree of precision.) Shake mixt. 5 min and let 
sep. Centrf., if necessary. Draw off and discard aq. phase thru 
glass capillary tube attached to vac. Wash isoamyl acetate phase 
with two 5 mL portions \N NaOH and one 5 mL portion IN 
HO. Shake 5 min after each addn, let layers sep., and draw 
off and discard aq. phase. Centrf. 2 min at 1500 rpm and mea- 
sure A or %T at 530 nm against isoamyl acetate. Det. Co from 
calibration curve relating A or log %T to Co content of std 
solns contg 0, 2, 4, and 5 (xg Co. 

Refs: Anal. Chem. 30, 1153(1958). JAOAC 48, 412(1965). 

CAS-7440-48-4 (cobalt) 



AOAC Official Methods of Analysis (1990) 



Other Elements 



31 



975.01 Copper in Fertilizers 

Atomic Absorption Spectrometry Method 
Final Action 

See 965.09. 



941.01* Copper in Fertilizers 

Long Volumetric Method 

Final Action 
Surplus 1970 



S*e 2.129-2.130, 11th ed. 



942.01 



Copper in Fertilizers 

Short Volumetric Method 

Final Action 1960 



A. Reagents 

(a) Sodium thiosulfate std soln. — 0.037V. Prep, daily by dilg 
0.17V soln, 942.27. 1 mL 0.037V Na 2 S 2 3 = 1.906 mg Cu. 

(b) Starch soln. — Mix ca 1 g sol. starch with enough cold 
H 2 to make thin paste, add 100 mL boiling H 2 0, and boil 
ca 1 min while stirring. 

(c) Bromocresol green indicator. — Dissolve 0.1 g terra- 
bromo-m-cresolsulfonphthalein in 1.5 mL 0.17V NaOH, and dil. 
to 100 mL with H 2 0. 

B. Determination 

Place 2 g sample in 300 mL erlenmeyer and add 10 mL 
HN0 3 and 5 mL H 2 S0 4 . Digest on hot plate to white fumes. 
If soln darkens, owing to org. matter, cool slightly, add little 
more HN0 3 , and digest again to white fumes, repeating op- 
eration if necessary until org. matter appears to be destroyed. 
Cool, add 50 mL H 2 0, boil ca 1 min, and cool to room temp. 

Add bromocresol green, then NH 4 OH until indicator changes 
to light green (pH 4). Cool again to room temp., and if in- 
dicator changes back to more acid color, add NH 4 OH dropwise 
until indicator becomes light green again, avoiding excess. Add 
2 g NH^Fj (toxic; see safety notes on toxic dusts), mix well, 
and let stand ca 5 min. Add 8-10 g KI, mix well, and titr. 
with std Na 2 S 2 3 soln to light yellow. Add ca 1 mL starch 
soln and continue titrg slowly until color is nearly same as just 
before addn of the Kl and becomes no darker on standing 20 
sec. Report as % Cu. 

Ref.: JAOAC 25, 77,352(1942). 

CAS-7440-50-8 (copper) 



(d) Mercuric chloride saturated soln. — Shake HgCl 2 with 
H 2 and let settle. 

(e) Stannous chloride soln. — Dissolve 20 g Sn0 2 .2H 2 in 
20 mL HC1, warming gently. Add 20 mL H 2 and dil. to 100 
mL with HC1 (1 + 1). Keep warm until clear; then add few 
granules Sn. Dispense from dropping bottle. 

B. Preparation of Sample Solution 

(a) Suitable for all fertilizers. — Treat 1 g as in 957.028(e), 
using 15 mL HCIO4. Hold =>1 hr at ca 170° to remove HN0 3 
completely. Dil. to 200 mL. 

(b) Suitable for soluble salts and oxides. — Dissolve 1 g in 
10 mL HC1, warming gently. Dil. to 200 mL. 

C. Reduction 

Heat aliquot of sample soln (100 mL and 50 mL, resp., for 
samples contg <0.5 and 0.5-4.0% Fe) to bp. Add few drops 
diphenylamine sulfonate soln; then SnCl 2 soln dropwise until 
violet color is discharged and 2 drops excess. (Usually 1-6 
drops are required. Larger amt may be used with samples contg 
large amt of Fe.) If reduction does not occur, discard and pro- 
ceed as follows with second aliquot: 

Add few granules Zn, boil few min, and either filter off 
excess Zn, washing with hot H 2 0, or let Zn dissolve. Heat to 
bp and finish reduction with SnCl 2 and diphenylamine sulfo- 
nate indicator as before. Add 10 mL HC1 (1 + 1). Adjust vol. 
to 75-110 mL with H 2 0. Cool rapidly in cold H 2 0. Add 10 
mL satd HgCl 2 soln, swirl gently, add 5 mL H 3 P0 4 , and titr. 
immediately. (Small amt of HgCJ must ppt to ensure complete 
reduction.) 

D. Titration 

Add I drop diphenylamine indicator by pipet (no more; ex- 
cess will interfere with end point if amt of Fe is small). Titr. 
with 0.0 17V K 2 Cr 2 7 soln. Since end point may be difficult to 
see with very small amt Fe, approach end point slowly, al- 
lowing few sec for color to develop. Titr. to permanent blue 
(sometimes green with very small amt Fe). For samples contg 
>4% Fe, use 0AN K 2 Cr 2 7 for titrn. 1 mL 0.17V K 2 Cr 2 7 = 
0.00558 g Fe; 1 mL 6.017V = 0.000558 g Fe. 

Ref.: JAOAC 50, 397(1967). 

CAS-7439-89-6 (iron) 



980.01 Iron in Fertilizers 

Atomic Absorption Spectrophotometric Method 
Final Action 

See 965.09. 



967.01 Iron in Fertilizers 

Titrimetric Method 

First Action 1967 
Final Action 1969 

(Note: Diphenylamine may be harmful. See safety notes on 
mercury salts and toxic dusts.) 

A. Reagents 

(a) Diphenylamine soln. — Dissolve I g in 100 mL H 2 S0 4 . 

(b) Diphenylamine sulfonate soln. — Dissolve 0.5 g in H 2 
in 100 mL vol. flask and dil. to vol. 

(c) Potassium dichromate std solns. — 0. IN and 0.017V. Prep. 
0AN K 2 Cr 2 7 as in 949.13C. Prep. 0.017V soln by dilg 100 
mL 0.17V soln to 1 L. 



983.03 Iron (Chelated) 

in Iron Chelate Concentrates 

Atomic Absorption Spectrophotometric Method 

First Action 1983 

(Applicable to Fe ethylenediaminetetraacetate (EDTA), Fe hy- 
droxy ethylethylenediaminetriacetate (HEDTA), Fe diethyl- 
enetriaminepentaacetate (DTPA), Fe ethylenediaminedi-o-hy- 
droxyphenylacetate (EDDHA), and Fe citrate. Not applicable 
to mixed fertilizers, or to samples contg non-chelated metals 
other than Fe.) 

A. Principle 

Sample is dissolved in H 2 0, and non-chelated Fe is pptd as 
FeOH 3 at pH 8.5 and removed. Chelated Fe is detd by AAS, 
using std solns contg Na 2 H 2 EDTA. 



32 



Fertilizers 



AOAC Official Methods of Analysis (1990) 



B. Apparatus and Reagents 

(a) Sodium hydroxide soln. — 0.5N. Dissolve 20 g NaOH 
in H 2 and dil. to 1 L. 

(b) Disodium EDTA soln.— 0. 66%. Dissolve 0.73 g 
Na 2 H 2 EDTA.2H 2 in H 2 0, and dil. to 100 mL. 

(c) Iron std solns. — (/) Stock soln. — 1000 jxg Fe/mL. Dis- 
solve 1 .000 g pure Fe wire in ca 30 mL 6N HC1 with boiling. 
Dil. to 1 L. (2) Intermediate soln. — 100 fxg Fe/mL. Pipet 10 
mL Fe stock soJn and 10 mL Na 2 H 2 EDTA soln into 100 mL 
vol. flask and dil. to vol. with H 2 0. (3) Working solns. — Dil. 
aliquots of intermediate soln with 0.5N HO to make >4 std 
solns within range of detn (2-20 |xg Fe/mL). 

(d) Atomic absorption spectrophotometer. — With air~C 2 H 2 
flame. See 965.09A(a). 

C. Determination 

Weigh sample contg ca 40 mg Fe into 200 mL tall-form 
beaker. Wet with 2-3 drops of alcohol and dissolve in 100 
mL H 2 0. Add 4 drops of 30% H 2 2 , mix and adjust pH of 
soln to 8.5 with 0.5N NaOH. If pH drifts above 8.8, discard 
soln and repeat analysis. Transfer soln to 200 mL vol. flask, 
dil. to vol. with H 2 Q, and mix. Filter soln thru quant, paper. 
Pipet 10 mL filtrate into 200 mL vol. flask and dil. to vol. 
with 0.5N HO. Measured of solns, using lean air-C 2 H 2 flame 
as in 965. 09D and det. concn of Fe in sample (jxg Fe/mL) 
from either calibration curve or digital concn readout. In same 
manner, det. Fe blank on all reagents used. 

% Chelated iron - (|xg Fe/mL in sample 

— fig Fe/mL in blank) X 0.4/g sample 

Refs.: J AOAC 66, 952(1983); 69, 280(1986). 



984.01 Magnesium (Acid-Soluble) 

in Fertilizers 
Atomic Absorption Spectrophotometry Method 
Final Action 



See 965.09. 



964.01 



Magnesium (Acid-Soluble) 

in Fertilizers 

EDTA Titration Method 

First Action 1964 
Final Action 1965 



(Applicable to samples contg <0.25% Mn or Zn) 

A. Reagents 

Use reagents 962.01A(a), (b), (c), (d), (f) (1 mL = 1 mg 
Ca, equiv. to 0.6064 mg Mg), (g), (h) (stdzd as in 964.01B), 
and in addn: 

(a) Triethanolamine soln . — ( 1 + 1 ) . 

(b) Potassium ferrocyanide soln. — Dissolve 4 g 
K 4 Fe(CN) 6 .3H 2 in 100 mL H 2 0. 

(c) Ferric ammonium sulfate soln. — Dissolve 136 g 
FeNH 4 (S0 4 ) 2 .12H 2 in H 2 contg 5 mL H 2 S0 4 , and diL to 1 
L. Filter if not clear. 

B. Standardization 

Pipet 10 mL Ca std soln into 300 mL erlenmeyer. Add 100 
mL H 2 0, 10 mL KOH-KCN soln, 2 drops triethanolamine soln, 
5 drops K 4 Fe(CN) 6 soln, and 15±1 mg caJcein indicator. Im- 
mediately place flask on mag, or other mech. stirrer in front 
of daylight fluorescent light and white background. While stir- 
ring, titr. with EDTA soln to disappearance of all fluorescent 



green and until soln remains pink. Titr. ^3 aliquots. From av., 
calc. Ca titer in mg/mL EDTA soln. Ca titer x 0.6064 = Mg 
titer in mg/mL. 

C. Preparation of Solution 

(Caution: See safety notes on wet oxidation and perchloric 
acid.) 

(a) Organic materials. — Weigh 1 g sample into 250 mL 
boiling flask or erlenmeyer. Add 5 mL HCl and 10 mL HN0 3 , 
and boil on hot plate or over low flame until easily oxidized 
org. matter is destroyed (ca 15 min). Cool, add 5 mL 70-72% 
HC10 4 , and heat to appearance of copious fumes and momen- 
tary cessation of boiling, but not to dryness. Cool, and transfer 
to 250 mL beaker with ca 100 mL H 2 0. Continue with pH 
adjustment, as in 964. 01D. 

(b) Inorganic materials and mixed fertilizers. — Weigh 1 g 
sample into 250 mL beaker. Add 5 mL HCl and 10 mL HNO3. 
Cover with watch glass and heat on asbestos mat on hot plate 
nearly to dryness (ca 30 min). If soln remains colored from 
org. residues, cool, add 5 mL HC10 4 (70-72%), and continue 
heating to copious fumes and momentary cessation of boiling, 
but not to dryness. 

D. Determination 

Cool prepd soln to room temp. Wash watch glass and inside 
of beaker to ca 100 mL with H 2 0. Using pH meter with glass 
electrode and mech. stirring, adjust to ca pH 3 with 30% KOH 
soln and finally to pH 4.0 with 10% KOH soln. Add 
FeNH 4 (S0 4 ) 2 soln, 5 mL for sample <7% P 2 5 , 10 mL for 
sample 7-15% P 2 5 , 15 mL for sample 16-30% P 2 5 , and 
proportionate amts for samples >30% P 2 5 . Adjust to pH 5.0 
with KOH solns as above, or with HCl (1+4) if pH is >5.0. 
Cool to room temp, and transfer to 250 mL vol. flask with 
H 2 0. Dil. to vol. with H 2 and mix. Let stand until ppt settles. 
Disturbing ppt as little as possible, filter enough soln for ali- 
quots required for titrn thru dry 1 1 cm fluted paper, Whatman 
No. 1 , or equiv. 

Pipet two equal aliquots contg <15 mg Ca + JVlg (usually 
25 mL) into two 300 mL erlenmeyers and dil. each to 100 mL 
with H 2 0. To one (titrn J for Ca + Mg) add 5 mL pH 10 
buffer soln, 2 mL KCN soln, 2 drops triethanolamine soln, 5 
drops K 4 Fe(CN) 6 soln, and 8 drops eriochrome black T indi- 
cator. Titr. immediately with EDTA soln, stirring and lighting 
as in stdzn. Color changes are wine red, purple, dark blue, to 
clear pure blue end point, becoming green if overtitrd. 

To second aliquot (titrn 2 for Ca) add 10 mL KOH-KCN 
soln, 2 drops triethanolamine soln, 5 drops K 4 Fe(CN) 6 soln, 
and 15 ±1 mg calcein indicator. Titr. immediately with EDTA 
soln as in stdzn. 

(Titrn 1 - Titrn 2) x Mg titer EDTA 

X 100/mg sample in aliquot = % Mg 

Titrn 2 x Ca titer EDTA 

x 10/mg sample in aliquot - % Ca 

Ref.: JAOAC 47, 450(1964). 

CAS-7439-95-4 (magnesium) 



937.01 * Magnesium (Acid-Soluble) 

in Fertilizers 

Gravimetric Method 
Final Action 
Surplus 1970 



See 2.123, 11th ed. 



AOAC Official Methods of Analysis (1990) 



Other Elements 



33 



940.01* Magnesium (Acid-Soluble) 

in Fertilizers 

Volumetric Method 

Final Action 
Surplus 1970 

See 2.124, 11th ed. 



937.02 Magnesium (Water-Soluble) 

in Fertilizers 
Final Action 1966 

(a) In potassium-magnesium sulfate, magnesium sulfate, and 
kieserite. — Weigh 1 g sample into 250 mL vol. flask, add 200 
mL H 2 0, and boil 30 min. Cool, dil. to vol. with H 2 0, and 
mix. If detn is to be conducted gravimetrically, 937.01*, or 
volumetrically, 940.01A*, see 2.125, 11th ed. 

(b) In other materials, including mixed fertilizers . — Weigh 
1 g sample into 500 mL vol. flask, add 350 mL H 2 0, and boil 
1 hr. Cool, dil. to vol., mix, and filter if necessary. If detn is 
to be conducted gravimetrically, 937.01*, or volumetrically, 
940.01A*, see 2.125, 11th ed. 

(c) By EDTA method. — Transfer aliquot soln prepd as in 
(a) or (b) to beaker and det. Mg as in 964. 01D, using HO or 
KOH to adjust pH. 

Refs.: JAOAC 20, 252(1937); 22, 270(1939); 23, 249(1940); 
24, 268(1941); 25, 326(1942). 



until brown fumes diminish; then boil 30 min. If org. matter 
is not destroyed, cool, add 5 mL HN0 3 , and boil. Repeat pro- 
cess until no org. matter remains, and boil until white fumes 
appear. Cool slightly, and add 50 mL H 3 P0 4 (1+9). Boil few 
min. Cool, dil. to 200 mL in vol. flask, mix, and let stand to 
allow pptn of CaS0 4 . 

Pipet 50 mL clear soln into beaker. Heat nearly to bp. With 
stirring or swirling, add 0.3 g KIO A for each 15 mg Mn pres- 
ent, and hold 30-60 min at 90-100°, or until color develop- 
ment is complete. Cool, and dil. to measured vol. that will 
provide satisfactory concn for colorimetric measurement by in- 
strument chosen (usually <20 ppm Mn). Compare in color- 
imeter against std KMn0 4 soln, or in spectrophtr at 530 nm. 
Calc. to Mn. 

Ref.: JAOAC 23, 249(1940). 

CAS-7439-96-5 (manganese) 



941.02* Manganese (Acid-Soluble) 

in Fertilizers 
Bismuthate Method 

Final Action 
Surplus 1970 

(Applicable to Mn"' 2 only) 
See 2.127-2.128, 11th ed. 



972.02 Manganese (Acid-Soluble) 

in Fertilizers 
Atomic Absorption Spectrophotometric Method 

First Action 1972 
Final Action 1974 

(a) Applicable to Mn +2 only. — Prep, sample soln as in 
940.02, omitting the 50 mL H 3 P0 4 (1+9). Proceed as in 
965.09D, using std solns prepd as in 965.09B(f) and (h), sub- 
stituting 0.5N H 2 S0 4 for 0.5/V HC1 in 965.09B(h). 

(b) Applicable to total Mn +2 and Mn A . — Prep, sample soln 
as in 965. 09C. Proceed as in 965. 09D, using std solns prepd 
as in 965.09(f) and (h). 

Ref.: JAOAC 55, 695(1972). 



940.02 Manganese (Acid-Soluble) 

in Fertilizers 
Colorimetric Method 
Final Action 

(Applicable to samples contg Mn +2 only and with <5% Mn) 

A. Reagent 

Potassium permanganate std soln. — 500 ppm Mn. Prep, and 
stdze as in 940.35, except use 1.4383 g KMn0 4 and 0.12 g 
Na oxalate. Transfer aliquot contg 20 mg Mn to beaker. Add 
100 mL H 2 0, 15 mL H 3 P0 4 , and 0.3 g KI0 4 , and heat to bp. 
Cool, and dil. to I L. Protect from light. Dil. this soln contg 
20 ppm Mn with H 2 (previously boiled with 0.3 g K10 4 /L) 
to make convenient working stds in range of concns to be com- 
pared . 

B. Determination 

Place 1 g sample in 200 mL wide-neck vol. flask or 250 
mL beaker. Add 10 mL H 2 S0 4 and 30 mL HN0 3 . Heat gently 



972.03 Manganese (Water-Soluble) 

in Fertilizers 
Atomic Absorption Spectrophotometric Method 

First Action 1972 
Final Action 1974 

(Applicable to Mn +2 only) 

Place 1 g sample in 50 mL beaker, wet with alcohol, add 
20 mL H 2 0, and let stand 15 min, stirring occasionally. Trans- 
fer to 9 cm Whatman No. 5 paper, and wash with small por- 
tions H 2 until filtrate measures ca 230 mL. Let each portion 
pass thru paper before adding more. Add 3-4 mL H 2 S0 4 to 
filtrate. Proceed as in 965. 09D, using std solns prepd as in 
965.096(f) and (h), substituting 0.5N fLS0 4 for 0.5N HC1 in 
965.09B(h). 

Ref.: JAOAC 55, 695(1972). 



974.01 



Sodium in Fertilizers 

Flame Photometric Method 

First Action 1974 



A. Reagents 

See 955.06*, and in addn: 

Sodium chloride. — Dry 2 hr at 105°. 

B. Preparation of Solution 

Prep, soln as in 955.06*, using 2.5 g sample (<4% Na) or 
1.25 g (4-20% Na). 

C. Preparation of Standard Curve 

(a) Samples containing 1% or more sodium. — Proceed as 
in 955.06*, using 1.271 1 g NaCl, range of diln 0-40 ppm Na, 
intervals <5 ppm, and full scale for 40 ppm Na. 

(b) Samples containing less than 1% sodium. — Proceed as 



34 



Fertilizers 



AOAC Official Methods of Analysis (1990) 



in 955.06*, using 1.2711 g NaCi, range of diln 0-10 ppm Na, 
intervals 2 ppm, and full scale for 10 ppm Na. 

D. Determination 

Transfer 25 mL (<4% Na) or 10 mL (4-20% Na) sample 
soln to 250 mL vol. flask, dil. to vol. with H 2 0, and mix (if 
internal std instrument is used, add required amt LiN0 3 before 
dilg to vol.). Atomize portions of sample several times to ob- 
tain reliable av. readings for each soln. Det. ppm Na from std 
curve (a) or (b). Calc. % Na as follows: 

0-4%: ppm Na/10 = % Na 
4-20%: ppm Na/2 = % Na 

Refs.: JAOAC 55, 986(1972); 56, 859(1973); 57, 1402(1974). 

CAS-7440-23-5 (sodium) 

983.04 Sodium in Fertilizers 

Atomic Absorption Spectrophotometry Method 
First Action 1983 

A. Reagents and Apparatus 

(a) Ammonium oxalate soln. — Dissolve 40 g (NH 4 ) 2 C 2 04 in 
1 L H 2 0. 

(b) Sodium chloride .—Dry 2 h at 105°. 

(c) Atomic absorption spectrophotometer . — Model AA6 
(replacement model is Spectr-AA Series, Varian Instrument 
Group), or equiv. 

B. Preparation of Solution 

Weigh 2.5 g (<4% Na) or 1.25 g (4-20% Na) sample into 
250 mL vol. flask, add 125 mL H 2 and 50 mL (NH^QAt 
soln, and boil 30 min. Cool, dil. to vol., mix, and pass thru 
dry filter. For samples contg <1% Na, use this soln for detn. 
For samples contg 1-20% Na, place 20 mL in 100 mL vol. 
flask, dil. to vol. with H 2 0, and mix. 

C. Preparation of Standard Curve 

Dissolve 2.5421 g dried NaCl in H 2 and dil. to 1 L (1000 
ppm Na). Prep, std solns to cover range 0-200 ppm at inter- 
vals <20 ppm Na. 

D. Determination 

Set wavelength at 330.3 nm using air-C 2 H 2 flame. Aspirate 
stds and samples. Plot curve from std values and det. Na con- 
tent of sample solns from plot of A against ppm Na. Calc. % 
Na as follows: 

<1%: ppm Na X 25 /M = %Na 
1-20%: ppm Na X 125/M = %Na 

where M = wt of sample (mg) . 

Ref.: JAOAC 66, 1234(1983). 

CAS-7440-23-5 (sodium) 



980.02 Sulfur in Fertilizers 

Gravimetric Method 

First Action 1980 
Final Action 1985 

(Caution; See safety notes on sodium hydroxide and perox- 
ides.) 

(a) Total sulfur (sulfate and elemental) in dry fertilizers. — 
Accurately weigh sample contg 100-150 mg S into 400 mL 
beaker, add 200 mL H 2 0, 15 mL HC1, heat to bp, and boil 



gently ca 10 min. Filter thru gooch crucible contg glass fiber 
paper and wash with hot H 2 0. Set washed crucible aside. 

Quant, transfer filtrate back to beaker and bring nearly to 
bp. Add slowly, with const stirring, slight excess (ca 15 mL) 
10% BaCL soln. Digest on low temp, hot plate adjusted so 
that soln does not boil, or on steam bath 1 hr, and let stand 
at room temp, overnight. Filter thru gooch crucible contg glass 
fiber paper previously dried at 250°, cooled, and weighed. Wash 
with ten portions hot H 2 0, dry crucible and contents 1 hr at 
250°, cool to room temp., and weigh. 

% Sulfate S = g BaS0 4 x 0.1374 x 100/g sample 

Wash insol. residue with five 10 mL portions acetone satd 
with S, dry 1 hr at 100°, cool, and weigh. Wash residue with 
three 5 mL portions CS 2 , drain, dry 1 hr at 100°, cool in des- 
iccator, and weigh. Difference in wt = elemental S (S°). 

Perform blank on S° portion of method by weighing 5.0 g 
fertilizer sample contg no S° into 400 mL beaker and pro- 
ceeding as above, beginning "add 200 mL H 2 0, .... Set 
washed crucible aside." Omit sulfate S detn and continue, be- 
ginning "Wash insol. residue with five. ..." Correct differ- 
ence in wt (— elemental S) for blank found. 



%S° 
% Total S 



gS x 100 /g sample 
% Sulfate S + % S° 



(If S° is <5%, use 5 g sample and repeat S° portion of detn.) 

(b) Total sulfur (sulfate, sulfite, thiosuifate, and sulfide) in 
liquid fertilizers. — Accurately weigh sample contg 100-150 
mg S into 400 mL beaker. Add 50 mL H 2 0, 2 mL 50% NaOH, 
and 2 mL 30% H 2 2 . Cover with watch glass and reflux 1 hr, 
adding 1 mL increments H 2 2 (^5 mL total) as reaction sub- 
sides. After 1 hr, wash watch glass and remove. Dil. to ca 175 
mL with H 2 0, acidify with ca 10 mL MCI (1 + 1), and bring 
to bp. Proceed as in (a), beginning "Add slowly, with constant 
stirring, ..." 

% Total S = g BaS0 4 x 0.1374 x 100/g sample 

(c) Total sulfur in sulfur-coated urea and elemental sulfur 
formulations. — Accurately weigh sample contg 200-300 mg 
S into 125 mL g-s erlenmeyer, add ca 50 mL H 2 0, stopper, 
and shake vigorously 30 sec. Filter quant, with suction thru 
gooch crucible contg glass fiber paper and wash with H 2 0. 
Proceed as in (a), beginning "Wash insol. residue with five 
10 mL portions ..." 

% S° = g S x 100/g sample 

Refs.: JAOAC 63, 854(1980); 64, 420(1981). 

CAS-7704-34-9 (sulfur) 



942.02* Zinc in Fertilizers 

Gravimetric Method 

Final Action 1976 

Surplus 1976 

(For samples contg >0.1% Zn) 
See 2.138, 12th ed. 

942.03* Zinc in Fertilizers 

Colorimetric Method 

Final Action 1976 
Surplus 1976 

(For samples contg <4% Zn) 
See 2.139, 12th ed. 



AOAC Official Methods of Analysis (1990) 



Other Elements 35 



975.02 Zinc in Fertilizers 

Atomic Absorption Spectrophotometric Method 
Final Action 1976 

See 965.09. 



973.01 Zinc in Fertilizers 

Zincon Ion Exchange Method 

First Action 1973 
Final Action 1976 

(Clean all glassware with hot chromic acid or HN0 3 (1 + 1). 

Rinse thoroly with H 2 0. Caution: See safety notes on chromic 

and perchromic acids and nitric acid.) 

A. Reagents 

(a) Anion exchange resin. — 100-200 mesh, strong base, 
polystyrene alkyl quaternary amine, 7% cross linkage. 

(b) Zincon indicator. — Dissolve 0.12 g zincon (o-[[a~[(2~ 
hydroxy-5 -sulfophenyl)azo J benzyl idenejhydrazinoj benzoic acid, 
Na salt) (J.T. Baker Inc., No. X690) in 5 mL 0.37V NaOH 
and dil. to 100 mL with H 2 0. Prep, fresh weekly. 

(c) Hydrochloric acid solns.—(l) 0.5N.— Dil. 20 mL HC1 
to 500 mL with H 2 0. (2) 0.25/V.— Dil. 2 mL HC1 to 100 mL 
with H 2 0. (3) 0.005N. —DiL 2.5 mL HCI to 6 L with H 2 0. 

(d) Borate buffer soln. — pH 9.8. Dissolve 4 g H 3 B0 3 in 
140 mL H 2 0. Add 5 mL NH 4 OH by pipet and then dropwise 
to pH 9.8. Check daily. 

(e) Ammonium thiocyanate. — \M. Dissolve 0.76 g NH 4 CNS 
in 10 mL H 2 0. 

(f) Zinc std so Ins. — (7) Stock soln. — 1000 ppm. Dissolve 
1.000 g pure Zn metal in small amt HCI-HNO3 (1 + 1). Evap. 
to small vol., add 3 mL HCI, and heat. Dil. to 1 L with H 2 0. 
(2) Working soln. — 10 ppm. Dil. 10 mL stock soln to 1 L 
with H 2 0. 

(g) Sodium hydroxide soln. — 0.3N. Dissolve ca 1 .25 g NaOH 
in 100 mLH 2 0. 

B. Preparation of Resin Column 

Wash 12 g new resin in 250 mL beaker with H 2 until 
washings are neut. Introduce resin as slurry into 25 X 2.2 cm 
chromatgc tube with fritted glass disk and stopcock at bottom. 
Mark vol. levels on column at 10, 40, and 50 mL above packed 
resin and on 250 mL separator at 90 mL. (Keep resin wet and 
store under liq. when not in use.) Connect separator to top of 
column thru stopper. Attach inverted U-shaped glass dispen- 
sing tube to 250 mL vol. flask thru vented stopper or cork and 
connect with Zn-free plastic tubing to stopcock of column with 
stopcock grease. See Fig. 973.01. 

Mount reservoir (aspirator bottle or carboy) contg > 1 L 
0.005iV HCI high enough to effect back washing. Attach Zn- 
free tubing and pinch clamp. 

C. Flow Calibration 

Use sweep sec hand of watch or stopwatch to establish flow 
rates. Det. number drops/mL leaving dispensing tube. Re- 
move separator and vol. flask; drain and then backwash resin 
(see 973. 01E). Remove reservoir tubing, open stopcock, elute 
40 drops from dispensing tube, and measure vol. Use this fac- 
tor to convert 0.5 mL/min (required in Zn elution, 973. 01E) 
to drops /sec. 

D. Preparation of Sample 

Remove separator and elution tubing from column. Activate 
resin by draining column and adding 50 mL 0.5jV HCI. Drain 
column to 40 mL mark. 



Vented 




Fig. 973.01 — Apparatus for elution of resin column 



(a) Samples containing 0.14% or more zinc. — Dissolve 1 .000 
g well-ground sample in 10 mL HCI and 5 mL HN0 3 in 250 
mL beaker. Evap. to near dryness on hot plate. (Caution: Do 
not bake.) Redissolve residue in ca 40 mL 0.5/V HCI, boiling 
gently if necessary. Filter thru Whatman No. 41 paper into 100 
mL vol. flask. Thoroly wash residue and dil. filtrate to vol. 
with 0.5N HCI. Drain column to 10 mL mark. Tap column to 
pack resin. Pipet aliquot contg 0.7-0.8 mg Zn onto column. 
Elute sample soln at ca 5 sec/drop. 

(b) Samples containing less than 0.14% zinc. — Weigh, to 
nearest mg, sample contg 0.7-0.8 mg Zn into 250 mL beaker. 
Digest and filter sample and prep, column as in (a). Tightly 
attach open separator to column. Close stopcock. Transfer en- 
tire sample soln to separator, rinsing with two 10 mL portions 
0.5N HCI. Open stopcock. Elute sample soln at ca 5 sec /drop. 
Remove empty separator, rinse twice with 20 mL 0.5yV HCI, 
and add rinses to remaining soln in column. 

E. Elution of Zinc 

After sample soln passes thru resin, immediately rinse col- 
umn with 0.5N HO at ca 1.5 sec. /drop until 1 mL eluate gives 
clear, colorless soln with \M NH 4 SCN. If Fe +3 is present, soln 
will turn brown. Drain resin and backwash with 0.005N HCI 
from reservoir thru elution tubing, forcing out air bubbles from 
tubing and column. Simultaneously, tap resin into suspension 
as it is forced up. Close column stopcock when liq. reaches 
50 mL mark on column. Attach dispensing tube to vol. flask. 
Reopen stopcock, and raise flask until flow just stops. Con- 
tinue ht adjustment until a drop remains in equilibrium at tip 
of dispensing tube and neither rises nor falls. Secure flask. 
Attach open separator with H 2 seal to column and close stop- 



36 



Fertilizers 



AOAC Official Methods of Analysis (1990) 



cock. Add 240 mL 0.0057V HCJ to separator and reopen stop- 
cock. Open column stopcock until rate of ca 0.5 mL/min is 
sustained 10 min. If rate decreases, increase rate slightly until 
nearly const. Let elution continue overnight. Then, if >90 mL 
remains in separator, readjust rate as above and continue elu- 
tion to 90 mL mark. Finally, lower flask, fill to 250 mL mark 
at convenient rate from dispensing tip, and mix. Detach hose 
and separator from column, and drain all 3. Reactive resin, 
and stopper column as in 973. 01D. 

F. Determination 

Pipet 20 mL eluate into 50 mL vol. flask contg small piece 
litmus paper. Make alk. with 0.3JV NaOH, then just acidic 
with 0.257V HC1. Pipet in 2 mL more acid, 5 mL buffer, and 
3 mL zincon soln. DiL to vol. with H 2 0. Similarly prep. 0, 
1, 2, and 3 ppm std solns, using 0, 5, 10, and 15 mL std Zn 
working soln, resp. Using ppm std soln as blank, det. A at 
620 nm 15-45 min after zincon addn. Plot std curve of ppm 
against A . 

% Zn = (C X F)/W 

where C — ppm from std curve; W = g sample; and F = 
0.0625 for samples contg <0. 14% Zn or 6.25/mL aliquot pi- 
petted onto resin for samples contg >0.14% Zn. 

Ref.: J AOAC 56, 846(1973). 
CAS-7440-66-6 (zinc) 



Table 983.05 Operating Parameters 



936.01* 



Acid-Forming 



or Nonacid-Forming Quality of Fertilizers 

Final Action 
Surplus 1970 

See 2.141-2.142, 11th ed. 



983.05 Aluminum in Aluminum 

Sulfate-type Soil Acidifiers 
Atomic Absorption Spectrophotometric Method 
First Action 1983 

A. Apparatus and Reagents 

(a) Atomic absorption spectrophotometer. — Perkin-Elmer 
Model 303, or equiv. See 972.06A for typical operating pa- 
rameters. 

(b) Diluting soln.— Add 20 mL H 2 S0 4 and 2.5 g NaCl to 
500 mL H 2 0. Dil. to J L with H 2 0. 

(c) Aluminum std solns. — (I) Stock soln. — 1 mg Al/mL 
(1000 ppm). Accurately weigh 1.000 g pure Al and dissolve 
in ca 25 mL HC1. Evap. almost to dryness, add 500 mL FLO, 
20 mL H 2 S0 4 , and 2.5 g NaCl, and dil. to 1 L with H 2 0. (2) 
Working solns. — Dil. aliquots of stock soln with dilg soln, 
(b), to make 4 std solns within range 50-150 ppm. 

B. Preparation of Sample 

Accurately weigh ca 1 g sample into 500 mL screw-cap er- 
lenmeyer, add 250 mL H 2 0, and shake on wrist-action shaker 
ca 15 min. Quant, transfer to 500 mL vol. flask and dil. to 
vol. with H 2 0. Filter thru Whatman No. 2 paper. Dil. aliquot 
as necessary (diln factor - F) with dilg soln, (b), for concn 
range 50-150 ppm. 

C. Determination 

Set up app. as shown in Table 983.05, or use previously 
established optimum settings for app. Zero app. while aspir- 
ating dilg soln, (b). Det. A of std solns within 50-150 ppm 



Wavelength, nm 
Slit width, mm 


309.3 
1 


Source, ma 


30 


N 2 0, aspirating 

N 2 0, auxiliary and aspirating 

C 2 H 2 fuel 

Flame 

Ht, burner to light path, in. 

Sample uptake, mL/min 

Optimum concn range, ^g/mL 


4.5 (scale divisions) 

5.5 (scale divisions) 

6 (metal ball scale division) 

reducing 

4 
50-150 



range, alternating with sample soln readings. Flush burner with 
dilg soln, (b), and check zero point between readings. 
Det. Al content from std curve of A against \xg Al/mL. 

% Al - (pig Al/mL) x F x (500/g sample) x 10" 4 

Ref.: JAOAC 66, 946(1983). 

CAS-7429-90-5 (aluminum) 



PEAT 

973.02 Sampling of Peat 

Procedure 1973 

(Moss, humus, and reed-sedge types) 

Use slotted single or double tube or slotted tube and rod, 
all with pointed ends and min. 1" diam. for loose materials. 
Use cutting type core sampler, with plunger, for compressed 
materials. Pennsylvania State Forage Sampler (NASCO, 901 
Janesville Ave, Fort Atkinson, WI 53538) is satisfactory core 
sampler. 

Take representative sample from lot or shipment as follows: 

(a) Packaged or baled peats. — Lay bag or bale horizontally 
and remove core diagonally from end to end. From lots of 1- 
10 bags, sample all bags; from lots of ^11, sample 10 bags. 
Take 1 core from each bag sampled; except for lots of 1-4 
bags, take diagonal cores from each bag and addnl cores to 
total >5 cores. 

(b) Bulk samples. — Draw >]0 cores from different re- 
gions. 

(c) Small containers (10 lb or less). — Take entire package. 
Working rapidly to prevent moisture losses, reduce composite 
sample to <500 g (by wt) or 2 L (by vol.) by mixing on clean 
plastic or paper and quartering. Place sample in air-tight con- 
tainer. 

Sampling by random "grab" procedure is necessary if par- 
ticle size range is to be detd or if representative sample cannot 
be taken with core sampler as above. 

Refs.: Book of ASTM Stds (1971) Pts 11, 22, and 30, ASTM 
D2973-D2978, D2980, and D2944. JAOAC 56, 

154(1973). 



967.02 Preparation of Peat Sample 

First Action 1967 
Final Action 1978 

Place representative field sample on square rubber sheet, pa- 
per, or plastic. Reduce sample to amt required by quartering 
and place in moisture-proof container. Work rapidly to prevent 
moisture losses. 



Ref.: JAOAC 50, 394(1967). 



AOAC Official Methods of Analysis (1990) 



Peat 37 



967.03 



Moisture in Peat 

First Action 1967 
Final Action 1978 



A. Method J 

Mix sample thoroly and place 10-12 g in ignited and weighed 
(with fitted heavy-duty Al foil cover) Vycor or porcelain evapg 
dish, ^75 mL capacity. Crush soft lumps with spoon or spat- 
ula. Cover immediately with Al foil cover and weigh to nearest 
mg. Dry, uncovered, 16 hr at 105°. Remove from oven, cover 
tightly, cool, and weigh. 

% Moisture (report to nearest 0.1%) 

= (g as-received sample - g oven-dried sample) 

X 100/g as-received sample 

B. Method II 

(Use when pH, N, fiber, etc., are to be detd.) 

Mix thoroly and weigh 100-300 g representative sample, 
967.02, and spread evenly on large flat pan. Crush soft lumps 
with spoon or spatula and let come to moisture equilibrium 
with room air >24 hr. Stir occasionally to maintain max. air 
exposure of entire sample. When wt is const, calc. loss in wt 
as % moisture removed by air drying. Grind representative 
portion air-dried sample 1-2 min in high-speed blender; use 
for moisture, ash, and N detns. 

Mix air-dried, ground sample and weigh, to nearest mg, 
equiv. of 10 g sample on as-received basis (g air-dried sample 
equiv. to 10.0 g as-received sample = 10.0 — 1(10.0 X % 
moisture removed)/ 100]). Place weighed sample in ignited and 
weighed (with fitted heavy duty Al foil cover) Vycor or por- 
celain evapg dish and proceed as in 967. 03 A. 

% Moisture (report to nearest 0.1%) 

- (10.0 - g oven-dried sample) X 10.0 

Ref.: J AOAC 50, 394(1967). 



973.03 Particle Size Range of Peat 

Mechanical Analysis 
First Action 1973 

A. Apparatus 

Mechanical sieve shaker. — With 8" diam., Nos. 8 and 20 
sieves equipped with cover and bottom pan. 

B. Preparation of Sample 
Air-dry as in 967. 03B. 

C. Determination 

Mix thoroly and place 20.0 g air-dried sample on No. 8 
sieve nested on No. 20 sieve. Secure sieves and shake at suit- 
able speed 10 min. Remove and weigh foreign matter, such 
as sticks, stones, and glass, from No. 8 fraction. Weigh frac- 
tions of peat retained on Nos. 8 and 20 sieves and portion 
collected in bottom pan. Convert fraction and sample wts to 
as-received basis and calc. in terms of %. (If foreign matter 
is absent, conversion to as-received basis is not necessary.) 

% Foreign matter = 

fraction removed from No. 8 sieve x 100 
% Coarse fiber = fraction retained on No. 8 sieve x 100 
% Medium fiber = fraction retained on No. 20 sieve x 100 
% Fines = fraction collected in pan X 100 

If mech. sieve shaker is not available, use hand sieving. 
Conduct sieving by appropriate lateral and vertical motions ac- 



companied by jarring action. Continue until no appreciable 
change is noted in sieve fraction. 

Refs.: Book of ASTM Stds(1971)Pts 11, 22, and 30, ASTM 
D2973-D2978, D2980, and D2944. JAOAC 56, 

154(1973). 



973.04 



pH of Peat 



A. Apparatus and Reagents 

(a) pH meter. — Battery-operated or on elec. line with volt- 
age regulator. 

(b) Carbon dioxide-free water. — See 964.24. 

(c) Acid potassium phthalate buffer soln.— 0.05m. See 
964.24(c). 

(d) Phosphate buffer soln.— 0.025m. See 964.24(d). 

(e) Calcium chloride solns (Method II only). — (7) Stock 
soln. — \.0M. Dissolve 147 g CaCl 2 .2H 2 in H 2 in 1 L vol. 
flask, cool, dil. to vol., and mix. Dil. 15 mL of this soln to 
200 mL with H 2 in vol. flask and stdze by titrg 25 mL aliquot 
dild soln. with std 0AN AgN0 3 , 941. 18C, using 1 mL 5% 
K 2 CrC 4 as indicator. (2) Working soln.—OMM (pH 5.0-6.5). 
Dil. 20 mL stock soln. to 2 L with H 2 0. 

S. Determination 

(a) Method I {in distilled water). — Weigh ca 3.0 g air-dried 
peat or equiv. amt moist material into 100 mL beaker. Add 
50 mL H 2 0. (Addnl H 2 may be needed for very fibrous ma- 
terials such as sphagnum moss peat.) Let soak 30 min, with 
occasional stirring. Read on pH meter. 

(b) Method II (in 0.0IM calcium chloride soln). — Weigh 
ca 3.0 g air-dried peat or equiv. amt moist material into 100 
mL beaker. Add 50 mL 0.01M CaCl 2 . Let soak 30 min, with 
occasional stirring. Read on pH meter. Report results as pH 
in 0.0 1M CaCl 2 soln. (pH values in CaCl 2 soln are usually ca 
0.5-0.8 units lower than those in H 2 0. Observed pH in CaCl 2 
soln is virtually independent of initial amt salt present in soil, 
whereas pH readings in H 2 can be modified by salts such as 
fertilizer material.) 

Refs.: Book of ASTM Stds(1971)Pts .11, 22, and 30, ASTM 
D2973-D2978, D2980, and D2944. JAOAC 56, 
154(1973). 



967.04 



Ash of Peat 

First Action 1967 
Final Action 1978 



Place uncovered (retain cover for weighing) Vycor or por- 
celain dish contg dried sample from moisture detn in furnace. 
Gradually bring to 550° and hold until completely ashed. Cover 
with retained Al foil cover, cool, and weigh. 

% Ash (report to nearest 0. 1%) = g ash X 100/g as-received 
sample taken for moisture detn. (If moisture Method 11 was 
used, g as-received sample - 10.0.) 

Ref.: JAOAC 50, 394(1967). 



973.05 Sand in Peat 

First Action 1973 

A. Preparation of Sample 
Air-dry as in 967.03B. 

B. Determination 

(Caution: See safety notes on chloroform.) 



38 



Fertilizers 



AOAC Official Methods of Analysis (1990) 



Place 25 g air-dried, ground sample into 125 mL tall-form 
beaker, or equiv. Nearly fill beaker with CHCI3, stir briefly, 
and let settle ca 1 min. With spoon, discard most floating org. 
material, decant remaining org. material and CHC1 3 , taking 
care not to disturb settled portion (sand), and air-dry to remove 
residual CHC1 3 . (Stirring aids drying.) 

When dry, weigh settled portion and calc. as % sand (in- 
cludes other minerals present such as limestone, etc.). 

% Sand = (g air-dried settled residue 

x 100)/g air-dried sample 

Refs.: Book of ASTM Stds (1971) Pts 11, 22, and 20, ASTM 
D2973-D2978, D2980, and D2944. JAOAC 56, 

154(1973). 



967.05 Organic Matter in Peat 

First Action 1967 
Final Action 1978 

% Org. matter = 100.0 - (% moisture + % ash) 
Ref.: JAOAC 50, 394(1967). 



973.06 Nitrogen (Total) in Peat 

First Action 1973 

Det. N as in 955.04, using well mixed, air-dried, ground 
sample equiv. to 10.0 g sample on as-received basis. 

Det. g air-dried sample equiv. to 10.0 g as -received sample 
as in 967.03B 

Refs.: Book of ASTM Stds (1971) Pts 1 1, 22, and 30, ASTM 
D2973-2978, D2980, and D2944. JAOAC 56, 

154(1973). 



969.05 Water Capacity and Volumes 

for Peat 

First Action 1969 
Final Action 1978 

A. Apparatus 

Dispensing apparatus. — 2 dispensing burets, 250 mL in 1 
mL subdivisions, ±2 mL tolerance, pinchcock type; 1-hole 
No. 6 rubber stopper; straight polyethylene drying tube with 
serrated rubber tubing fittings, 15 cm long, 3 / 4 " od, 5 /s" id (No. 
14782-2, Cenco, Inc., 2600 Kostner Ave, Chicago, IL 60623); 
and stainless steel screen circle, ca 16 mesh and 28.7 mm diam. 

Assemble dispensing app. as follows: Discard serrated rub- 
ber tubing fittings from polyethylene drying tube and use tube 
only. Center stainless steel screen on one end of tube and seal. 
(Soldering iron is useful.) Adjust length of tube to match con- 
venient graduation of buret; then scallop end without screen 
to allow for H 2 drainage, and insert into dispensing buret 
with screen side up. 

B. Preparation of Sample 
See 967.02. 

C. Determination 

Det. moisture content on sep. sample by 967. 03A or B. 

Weigh buret fitted with plastic tube and screen. Working 
rapidly to prevent moisture losses, mix sample thoroly, place 
on top of No. 4 screen, and shake until sieving is complete. 
Use only portion that has passed thru sieve for detn. Firmly 
pack buret with 25 cm (10") of 4 mesh sample as follows: 



Attach rubber stopper to delivery end of buret. Add ca 20 mL 
portions, firmly tapping 3 times vertically from ht of 15 cm 
(6") on rubber stopper, for final ht of 25 cm. (This will ensure 
that ht of final wet vol. is 19-25 cm.) Remove stopper; weigh 
buret to nearest g. Position buret to use sink as drain. Place 
H 2 source (19 L (5 gal.) bottle) equipped with siphon device 
above level of buret. Connect clamped rubber tubing of siphon 
device to buret with glass tubing (ca 13 cm (5") long, con- 
stricted at one end) inserted into one-hole rubber stopper fitting 
tightly into top of buret. Attach rubber tubing with pinch clamp 
to delivery end of buret. Open both clamps and pass H 2 thru 
sample ^24 fir, maintaining water reservoir over sample at all 
times. (Moss-type samples may float but gradually settle as 
sample becomes wet.) After initial soaking, regulate H 2 flow 
thru column by adjusting screw clamp at delivery end of buret. 
(In-flow of H 2 should be ca equal to out- flow; flow of ca 1 
drop/sec is suitable.) When sample is super satd, close both 
clamps and let sample settle in H 2 ca 5 min. Top surface of 
sample should be as level as possible. 

Raise buret and replace rubber tubing on delivery end of 
buret with 250 mL dispensing buret filled with H 2 O s using 
rubber stopper for connection. Connect two burets tightly, with 
no air leaks. Remove siphon device and open outlet clamps 
of both burets to empty. (Suction created is equiv. to ca 38 
cm (15") H 2 0. Check for air leaks to ensure that std suction 
is exerted on sample. It is important to remove excess H 2 as 
described.) Measure ht of wet peat. Ht should be 19-25 cm. 
Record vol. in mL and weigh buret, plastic tube with screen, 
and wet peat to nearest g. 

Wet sample again as above >]. hr, drain by suction, record 
vol., and weigh. Repeat until consistent results are obtained. 

D. Calculations 

(a) Saturated Volume Weights, g/mL 
As-recd = g as-recd sample/mL wet vol. 

Oven-dried = g dried sample/mL wet vol., where g dried 
sample — g as-recd sample X [(100 — % moisture)/! 00] 
Wet = g wet sample/mL wet vol. 

(b) Water-Holding Capacity, % 
(1) Weight basis: 

As-recd 

= Kg wet sample — g as-recd sample) 

x 100]/g as-recd sample 
Oven-dried 

- [(g wet sample ~ g dried sample) 

x 100] /g dried sample 
(2) Volume basis: 
Water vol. 

= Kg wet sample — g dried sample) 

x 100]/(mL wet vol. x 1.0) 

(c) Dry Peat Volume, % 

Dry peat vol. = (g dried sample 

X 100)/(mL wet vol. X 1.5) 

(d) Air Volume, % 

Air vol. = 100 ~ (% water vol. + % dry peat vol.) 

Refs.: JAOAC 51, 1296(1968); 52, 384(1969). 



973.07 



Volume, Peat 

Alternative Method 

First Action 1973 



A. Principle 

Method consists of dividing particles of peat from original 
container by passing them thru 12.7 mm (0.5") sieve and al- 
lowing them to fall into vol. -measuring container. 



AOAC Official Methods of Analysis (1990) 



Peat 



39 



S. Apparatus 

(a) Sieve.— No. 7/ (12.7 mm). 

(b) Measuring box. — Steel or wood, bound with metal hav- 
ing one of the following sets of inner dimensions: (7) l /2 cu. 
ft. - 12 X 12 x 12" with line scribed 6" from bottom; (2) 3 / 4 
cu. ft. = 12 x 12 X 12" with line scribed 9" from bottom; (J) 
1 cu. ft. - 12 x 12 X 12" box, 2 cu. ft. = 16 X 16 base x 
13.5" ht, 5 cu. ft - 16 x 16 base x 33.75" ht. 

C. Determination 

(a) Loose peat. — Remove material from bag or container, 
pass it thru l / 2 " sieve, and place directly into measuring box. 
Pour contents from ca 60 cm (2') into measuring box. Det. 
contents of bag or container only once. Fill corners of mea- 
suring box by shaking with rotary motion, 1 rotation/sec for 
5 sec, without lifting box from floor or surface. When filled, 
level off by straightedge. Use ht of box to calc. vol. in cu. ft. 

(b) Baled peat.- — Vol. baled material = ht X area of base. 
Correct measurements for outside wrappers. Det. amt loose 
peat in bale by passing thru ] / 2 ff sieve and measuring amt loose 
peat, using 12 x 12 X 12" box as in (a). Report vol. of peat 
in cu. ft. 

Report total vol. of sieved peat from original container. 

Refs.: Book of ASTM Stds(1971)Pts II, 22, and 30, ASTM 
D2973-D2978, D2980, and D2944. JAOAC 56, 
154(1973). 



973.08 Volume Weight, Water-Holding 

Capacity, and Air Capacity of Water-Saturated 
Peat Materials 
First Action 1973 

A. Apparatus 

(a) Hollow spray nozzle. — Monarch F-97-W, nozzle No. 
4.6160 (Monarch Mfg. Works Inc., 2501 E Ontario St, Phil- 
adelphia, PA 19134), or equiv. 

(b) Pipe connection. — For installation of nozzle on H 2 
faucet in sink. 

(c) Containers. — Approx. 2 L (2 lb coffee cans are suita- 
ble) fitted with plastic covers. Replace metal bottom of one 
with No. 20 Cu screen (test container). 

(d) Aluminum pie pans. — 20 cm (8") diam. Drill holes in 
side walls of pan so that H 2 depth in pan remains ca 1.3 cm 
(0.5"). 

B. Preparation of Sample 
See 967.02. 

C. Determination 

Det. moisture content on sep. sample by 967. 03A or B. 

Weigh test container fitted with plastic cover, screen, and 
circle of filter paper (12.5 cm Whatman No. 4, or equiv.) which 
is placed on screen. Thoroly mix equal wts of H 2 and peat 
and place in container without pressure to ht of 10 cm (4"); 
record wt in g. (If peat is dried out, mix 1 part peat with 2 
parts H 2 0. If wet, mix 2 parts peat with 1 part H 2 0.) 

Place test container in Al pan filled with H 2 in sink >30 
cm (12") directly under spray nozzle. Water ca 24 hr as mist 
to prevent compression of peat. Place cover on container, seal 
(tape is suitable) to prevent evapn, and let stand in Al pan, 
maintaining 1.3 cm FLO head 2 days. Remove from pan and 
drain 2 hr with container at 45° angle. Remove seal on cover, 
and record wt and vol. Vol. can be detd by using container 
identical to test container not fitted with screen, filling H 2 to 



same ht as sample in test container, and transferring to grad- 
uate with mL markings. 

D. Calculations 

(a) Saturated Volume Weights, g/mL 

(1) As-recd = g as-recd sample/mL wet vol., where g as- 
recd sample = g total sample/2; or g total sample/3 if 2 parts 
H 2 used; or (g total sample X 2)/3 if 2 parts peat used. 

(2) Oven-dried.— See 969. 05D 

(3) Wet.— See 969.05D. 

(b) Water -Holding Capacity, %.—See 969.051). 

(c) Dry Peat Volume, %.—See 969.05D. 

(d) Air Volume, %.—See 969. 05D 

Refs.: Book of ASTM Stds(1971)Pts 11, 22, and 30, ASTM 
D2973-D2978, D2980, and D2944. JAOAC 56, 

154(1973). 



973.09 Cation Exchange Capacity for Peat 
Titrimetric Method 

First Action 1973 

Final Action 1978 

AOAC-ASTM Method 

A. Principle 

Cation exchange capacity is measure of total amt exchange- 
able cations that can be held by peat, expressed as mequiv./ 
100 g air-dried peat. Peat sample is shaken with 0.5/V HC1 to 
remove bases and to sat. sorption complex with H + . Excess 
acid is removed; absorbed H + is replaced with Ba H " 2 , titrd with 
O.WNaOH, using phthln indicator, and calcd to mequiv./lOO 
g air-dried peat. 

B. Reagents 

(a) Dilute hydrochloric acid. — 0.5/V. Dil. 42 mL HC1 to 1 
L with H 2 0. 

(b) Barium acetate soln. — 0.5/V. Dissolve 64 g Ba(OAc) 2 
in H 2 and dil. to 1 L. 

(c) Silver nitrate soln. — 1%. Dissolve 1 g AgN0 3 in 100 
mL H 2 0. 

(d) Sodium hydroxide std soln.— 0. IN. Prep, and stdze as 
in 936.16 

C. Preparation of Sample 
See 967.02. 

D. Determination 

Thoroly mix air-dried ground peat sample and place 2.00 g 
in 300 mL erlenmeyer. Add ca 100 mL 0.57V HC1; stopper 
flask and shake vigorously periodically during 2 hr (or shake 
mech. 30 min). Filter thru rapid paper (24 cm fluted, or equiv.) 
in large powder funnel. Wash with 100 mL portions H 2 until 
10 mL wash shows no ppt with ca 3 mL 1% AgN0 3 . Discard 
filtrate. Immediately transfer moist peat to 300 mL erlen- 
meyer, by puncturing apex of paper and forcing moist peat 
thru funnel stem into erlenmeyer, using spray from wash bottle 
contg ca 100 mL 0.5/V Ba(OAc) 2 . Stopper flask and shake vig- 
orously periodically during 1 hr (or shake mech. 15 min). Fil- 
ter, and wash with three 100 mL portions H 2 0. Discard peat, 
and titr. washings with 0, IN NaOH, using 5 drops phthln, to 
first pink. 

Calc. mequiv./ 100 g air-dried peat 

= (mL X normality NaOH x 100)/g sample. 

Ref.: JAOAC 56, 154(1973). 



3* Plants 

Robert A- Isaac, Associate Chapter Editor 

University of Georgia 



922.01 



Sampling of Plants 
Final Action 



When more than one plant is sampled, include enough plants 
in sample to ensure that it adequately represents av. compo- 
sition of entire lot of plants sampled. (This number depends 
upon variability in composition of the plants.) Det. details of 
sampling by purpose for which sample is taken. 

Refs.: Botan. Gaz. 73, 44(1922). Proc. Am. Soc. Hort. Sci. 
1927, p. 191. JAOAC 13, 224(1930); 16, 71(1933); 
19, 70(1936). 



922.02 



Plants 

Preparation of Sample 

Final Action 



(a) For mineral constituents . — Thoroly remove all foreign 
matter from material, especially adhering soil or sand, but to 
prevent leaching, avoid excessive washing. Air- or oven-dry 
as rapidly as possible to prevent decomposition or wt loss by 
respiration, grind, and store in tightly stoppered bottles. If re- 
sults are to be expressed on fresh wt basis, record sample wts 
before and after drying. When Cu, Mn, Zn, Fe, Al, etc. are 
to be detd, avoid contaminating sample by dust during drying 
and from grinding and sieving machinery. 

(b) For carbohydrates . — Thoroly remove all foreign matter 
and rapidly grind or chop material into fine pieces. Add weighed 
sample to hot redistd alcohol to which enough pptd CaC0 3 has 
been added to neutze acidity, using enough alcohol so that 
final concn, allowing for H 2 content of sample, is ca 80%. 
Heat nearly to. bp on steam or H 2 bath 30 min, stirring fre- 
quently. (Samples may be stored until needed for analysis.) 

Refs.: Botan. Gaz. 73, 44(1922). Proc. Am. Soc. Hort. Sci. 
1927, p. 191. JAOAC 13, 224(1930); 16, 71(1933); 
19, 70(1936). 



930.04 



Moisture in Plants 
Final Action 1965 



See 934.01, 920.36*, or 930.15. 



930.05 



Ash of Plants 
Final Action 1965 



See 900.02A, 900.02B, or 942.05. 

920.08* Sand and Silica in Plants 

Gravimetric Method 

Final Action 
Surplus 1989 

See 3.005, 14th ed. 



METALS 



953.01 Metals in Plants 

General Recommendations 
for Emission Spectrographs Methods 

Final Action 1988 

(Applicable to aluminum, barium, boron, calcium, copper, iron, 
magnesium, manganese, molybdenum, phosphorus, potas- 
sium, sodium, strontium, and zinc) 

(a) Instrumental technic. — If, because of equipment limi- 
tations, described methods cannot be followed in detail, or if 
detn of other elements is desired, following protocol is rec- 
ommended: Det. experimentally, with available facilities, po- 
tentials of various sample prepns and excitation conditions with 
relation to element detectability and general concn require- 
ments. Select analysis lines on basis of desirable intensity and 
freedom from spectral interference by other elements, as detd 
by prepg spectrum of each component element at av. concn at 
which it occurs in samples to be analyzed. Line and phototube 
characteristics are usually detd by instrument manufacturer. 

(b) Precision. — Stdze all conditions of technic and det. re- 
producibility of results by making ca 20 successive exposures 
on sample of representative composition. For each element, 
calc. std deviation of single exposure and divide by square root 
of number of individual exposures that will be averaged in 
practice to constitute 1 detn. From this est. of std deviation of 
single detn, calc. coefficient of variation for each element. 
Following upper limits for precision error of spectrographic 
detns in analysis of plant material are satisfactory in relation 
to other routine methods or to practical requirements: Coef- 
ficients of variation (%) — Ca, Mg, Mn, and Mo, 3-7; B, Ba, 
Cu, K, P, and Zn, 7-15; and Al, Fe, and Na, >15. Coeffi- 
cients of variation vary from instrument to instrument for each 
element; above values were obtained from std plant tissue by 
11 different instruments. 

(c) Accuracy. — Precise technic is essential but not only fac- 
tor involved in accuracy. Reliability and appropriateness of stds 
and judgment used in ref. method are of utmost importance. 
Failure in any of these respects can result in serious calibration 
error for otherwise satisfactory method. 

Carefully prep, synthetic stds from highest grade H 2 0-free 
analyzed chems, collectively blanked for minor and trace ele- 
ments. Preferably confirm values assigned to natural stds by 
results of >1 laboratory. 

Matrix similarity between stds and samples, or closely con- 
trolled correction system for matrix differences, is essential. 
Check correction scales frequently against stds which closely 
match particular types of plant materials being analyzed. 

Precision error of technic applies to ref. exposures as well 
as to samples. For this reason, base fiducial adjustments on as 
many ref. exposures as may be feasibly included in each series 
of samples. 

Refs.: JAOAC 36, 41 1.(1953); 37, 721(1954); 58, 764(1975). 



40 



AOAC Official Methods of Analysis (1990) 

980.03 Metals in Plants 

Direct Reading Spectrographic Method 
Final Action 1988 

A. Apparatus 

(a) Spark excitation source. 

(b) Spectrograph. — 1.5 m grating spectrograph with spark 
stand and disk attachment rotating at 30 rpm. 

(c) Electrode sharpener. 

(d) Disk electrode. — High purity graphite disk 0.492" diam. 
and 0.200" thick. 

(e) Upper (pin) electrode. — Point appropriate lengths of std 
grade spectrographic C rods, 0.180" diam., in pencil sharpener 
equipped with pin stop to produce Vig" diam. flat tip. 

(f) Porcelain boat. — 60 mm long, 10 mm wideband 8 mm 
high (Coors No. 2, or equiv.). 

B. Reagents 

(a) Buffer.— Dissolve 50 g Li 2 C0 3 in 200 mL HN0 3 , and 
dil. to 1 L with H 2 0. (Caution: See safety notes on nitric acid.) 

(b) Element stock std solns. — On basis of expected sample 
concn range, prep, stock solns from individual pure nitrates, 
chlorides, or carbonate salts, or metal of resp. elements, as 
indicated in Table 980.03A. 

(c) Mixed element std solns. — Prep. 5 std solns contg % or 
ppm element indicated in Table 980. 03B as follows: Dissolve 



Metals 



41 



50 g Li 2 C0 3 in 200 mL HN0 3 , pipet in indicated aliquots, and 
dil. to 1 L with H 2 0. Prep, fresh every 6 months. 

C. Preparation of Sample 

Dry plant material 24 hr at 80° and grind in Wiley mill with 
No. 20 stainless steel sieve. Store in air-tight containers or in 
coin envelopes in dry atm. 

Weigh 1.0 g prepd sample into 30 mL high form crucible 
(porcelain is satisfactory). Ash >4 hr at 500° with crucible 
resting on asbestos plate rather than on floor of furnace. Cool, 
add 5.0 mL buffer soln, (a), stir, and let stand 30 min. 

D. Determination 

(a) Excitation. — Align and space electrodes 4 mm apart in 
holders; position pin electrode over disk electrode. Set source 
parameters to give uniform breakdown voltage at tandem air 
gap with operating parameters at 4 breaks/cycle and 4 amp 
(parameters may vary with source for best operating effi- 
ciency). 

Place aliquot of prepd soln in porcelain boat, set boat on 
arc stand, and raise to immerse Vie" of disc in soln. Spark 10 
sec to condition electrodes and photomultiplier tubes and then 
spark addnl 30 sec for integration. 

(b) Calibration. — Calibration technic varies with instru- 
ment. Use mixed element std solns and known plant tissue stds 
to calibrate spectrograph by same technic as for samples. Prep, 
std curves to cover desired concn range, using ratio to internal 
std and background correction for best results. 



Table 980.03A Preparation of Stock Standard Solutions 



Element Salt 


Element, 
9/L 


Salt, 

g/L Solvent 


955.08* 


Metals in Plants 




K KCI 


125 


238.36 H 2 
99.89 1 N HNO3 




Direct Current Arc Excitation Method 


Ca CaC0 3 


40 




Surplus 1970 




Mg MgO 


20 


33.16 IA/HNO3 








P H 3 P0 4 


10 


31 .64 H 2 


See 44.003-44.006. 11th ed. 




Na NaCl 


10 


25.42 H 2 








Fe Fe metal 


1 


1.00 IA/HNO3 








Mn MnO 


1 


1.29 1A/HN0 3 








Al Al metal 


1 


1.00 1 N HN0 3 








Zn Zn metal 


1 


1 .00 "\N HNO3 


955.09* 


Metals in Plants 




Cu Cu metal 


1 


1 .00 1 N HNO3 








Ba BaCI 2 


1 


1 .52 H 2 




Alternating Current Spark Excitation Method 


Sr SrC0 3 


1 


1.68 IA/HNO3 




Surplus 1970 




B H3BO3 


1 


5.72 H 2 








Mo (NH 4 ) 6 Mo 7 24 .H 2 


0.1 


1.29 H 2 


See 44.007-44.011, 11th ed. 




Table 980.03B Preparation of Mixed Element Standard Solutions 










Element, g/L 






Standard Solution Number 




Element 


1 


2 


3 4 


5 










mL to give (%) a 




K (CAS-7440-09-7) 


125 


2(0.5) 


4(1.0) 


8(2.0) 12(3.0) 


20(5.0) 


Ca (CAS-7440-70-2) 


40 


100(2.0) 


50(1.0) 


30(0.6) 10(0.2) 


15(0.3) 


Mg (CAS-7439-95-4) 


20 


100(1.0) 


70(0.7) 


50(0.5) 20(0.2) 


10(0.1) 


P (CAS-7723-14-0) 


10 


2(0.1) 


4(0.2) 


6(0.3) 10(0.5) 
mL to give (ppm) a 


14(0.7) 


Na (CAS-7440-23-5) 


10 


1(50) 


2(100) 


10(500) 20(1000) 


40(2000) 


Fe (CAS-7439-89-6) 


10 


10(500} 


4(200) 


2(100) 1(50) 


6(300) 


Mn (CAS-7439-96-5) 


10 


0.4(20) 


1(50) 


2(100) 4(200) 


10(500) 


Ai (CAS-7429-90-5) 


10 


0.6(30) 


1(50) 


2(100) 4(200) 


10(500) 


Zn (CAS-7440-66-6) 


1 


2(10) 


4(20) 


6(30) 10(50) 


20(100) 


Cu (CAS-7440-50-8) 


1 


1(5) 


2(10) 


4(20) 10(50) 


14(70) 


Ba (CAS-7440-39-3) 


1 


20(100) 


10(50) 


4(20) 2(10) 


1(5) 


Sr (CAS-7440-24-6) 


1 


40(200) 


20(100) 


10(50) 6(30) 


3(15) 


B (CAS-7440-42-8) 


1 


1(5) 


2(10) 


4(20) 10(50) 


14(70) 


Mo (CAS-7439-98-7) 


0.1 


2(1) 


4(2) 


8(4) 12(6) 


20(10) 



a Concn based on 1 g sample taken up in 5 mL buffer. 



42 



Plants 



AOAC Official Methods of Analysis (1990) 



975.03 Metals in Plants 

Atomic Absorption Spectrophotometry Method 

First Action 1975 
Final Action 1988 

(Applicable to calcium, copper, iron, magnesium, manganese, 
potassium, and zinc) 

A. Apparatus and Reagents 

Deionized H 2 may be used. See 965. 09 A and B, and fol- 
lowing: 

(a) Potassium stock soln. — 1000 |xg K/mL. Dissolve 1.9068 
g dried (2 hr at 105°) KC1 in H 2 and dil. to 1 L. Use fol- 
lowing parameters for Table 965.09: 7665 A, air-C 2 H 2 flame, 
and 0.04-2 |mg/mL range. 

(b) Calcium stock solas. — Prep. Ca stock soln and working 
stds as in 965.09B. 

(c) Cu, Fe, Mg, Mn, and Zn stock solns. — Prep, as in 
965.09B(b), (c), (e), (f), and (g). 

(d) Working std solns. — Dil aliquots of solns (c) with 10% 
HC1 to make > 4 std solns of each element within range of 
detn. 

B. Preparation of Sample 

(a) Dry ashing. — Accurately weigh 1 g sample, dried and 
ground as in 922.02(a), into glazed, high-form porcelain cru- 
cible. Ash 2 hr at 500°, and let cool. Wet ash with 10 drops 
H 2 0, and carefully add 3-4 mL HN0 3 (1 + 1). Evap. excess 
HN0 3 on hot plate set at 100-120°. Return crucible to furnace 
and ash addnl 1 hr at 500°. Cool crucible, dissolve ash in 10 
mL HC1 (1 + 1), and transfer quant, to 50 mL vol. flask. 

(b) Wet ashing. — Accurately weigh 1 g sample, dried and 
ground as in 922.02(a), into 150 mL Pyrex beaker. Add 10 
mL HNO3 and let soak thoroly. Add 3 mL 60% HCIO4 and 
heat on hot plate, slowly at first, until frothing ceases. (Cau- 
tion: See safety notes on wet oxidation.) Heat until HN0 3 is 
almost evapd. If charring occurs, cool, add 10 mL HN0 3 , and 
continue heating. Heat to white fumes of HCIO4. Cool, add 
10 mL HC1 (1 + 1), and transfer quant, to 50 mL vol. flask. 

C. Determination 

To soln in 50 mL vol. flask, add 10 mL 5% La soln, and 
dil. to vol. Let silica settle, decant supernate, and proceed as 
in 965.09D. 

Make necessary dilns with 10% HC1 to obtain solns within 
ranges of instrument. 

D. Calcuiations 

ppm Element = (|xg/mL) X F/g sample 
% Element = ppm x 10~ 4 

where F = (mL original diln x mL final diln)/mL aliquot if 
original 50 mL is dild. 

Ref.: JAOAC 58, 436(1975). 



985.01 Metals and Other Elements in Plants 

Inductively Coupled Plasma Spectroscopic Method 

First Action 1985 
Final Action 1988 

(Applicable to B, Ca, Cu, K, Mg, Mn, P, and Zn) 

A. Principie 

Sample is dry-ashed, treated with HNO3, and dissolved in 
HC1; elements are detd by ICP emission spectroscopy. 



B. Reagents and Apparatus 

(a) Stock solns. — 1000 |xg/mL. Weigh designated reagent 
into sep. 1 L vol. flasks, dissolve in min. amt of dissolving 
reagent, 



and dil. to vol. with H 2 0. 



Element 


Reagent 


g 


Dissolving Reagent 


B 


H3BO3 


5.7192 


H 2 


Ca 


CaC0 3 


2.4973 


6N HCI 


Cu 


pure metal 


1.0000 


HNO3 


K 


KCI 


1.9067 


H 2 


Mg 


MgS0 4 .7H 2 


10.1382 


H 2 


Mn 


Mn0 2 


1.5825 


6N HCI 


P 


NH 4 H 2 P0 4 


3.7138 


H 2 


Zn 


pure metal 


1.0000 


6N HCI 



(b) Std solns. — Pipet following vols of stock soln into 1 L 
vol, flasks. Add 100 mL HCI and dil. to vol. with H,0. 







Std Soln 1 




Std Soln 2 




Stock 


Final 


Stock 


Final 


Element 


Soln, m 


L Concn, M-g/ m L 


Soln, mL Concn, ^g/mL 


B 








10 


10 


Ca 


5 


5 


60 


60 


Cu 








1 


1 


K 


5 


5 


60 


60 


Mg 


1 


1 


20 


20 


Mn 








10 


10 


P 


5 


5 


60 


60 


Zn 








10 


10 



Make any needed subsequent dilns with 10% HCI (1 +9). 

(c) ICP emission spectrometer. — Suggested operating pa- 
rameters: forward power, 1.1 kilowatts; reflected power, <10 
watts; aspiration rate, 0.85-3.5 mL/min; flush between sam- 
ples, 15-45 s; integration time, 1-10 s. 



Element 


Wavelength, A 


B (CAS-7440-42-8) 


2496 


Ca (CAS-7440-70-2) 


3179 


Cu (CAS-7440-50-8) 


3247 


K (CAS-7440-09-7) 


7665 


Mg (CAS-7439-95-4) 


2795 


Mn (CAS-7439-96-5) 


2576 


P (CAS-7723-14-0) 


2149 


Zn (CAS-7440-66-6) 


2138 



C. Dry Ashing 

Accurately weigh 1 g sample, dried and ground as in 
922.02(a), into glazed, high-form porcelain crucible. Ash 2 h 
at 500°, and let cool. Wet ash with 10 drops of H 2 0, and care- 
fully add 3-4 mL HN0 3 (1 + 1). Evap. excess HNO a on hot 
plate set at 100-120°. Return crucible to furnace and ash addnl 
1 h at 500°. Cool crucible, dissolve ash in 10 mL HCI (1 + 
1), and transfer quant, to 50 mL vol. flask. Dil. to vol. with 
H 2 0. 

D. Determination 

Elemental detn is accomplished by inductively coupled plasma 
emission spectroscopy. Calibration of instrument is done thru 
use of known calibration stds. After calibration is complete, 
samples can be analyzed. Check calibration after every 10 
samples. If instrument has drifted out of calibration (>3% of 
original values), recalibrate. 

Calc. concn for each element of each dild sample as jxg/ 
mL. 

Ref.: JAOAC 68, 499(1985). 



AOAC Official Methods of Analysis (1990) 



Metals 



43 



INDIVIDUAL METALS 

928.03 Aluminum and Iron in Plants 

Titrimetric Method 
Final Action 

(Caution: See safety notes on sulfuric acid.) 

Take aliquot of Soln /, 920.08, contg enough Fe and Al to 
form ca 40 mg Fe- and A1P0 4 . Add few drops HN0 3 , Br-H 2 0, 
or H 2 2 to oxidize Fe. If soln does not already contain excess 
phosphate, add 0.5 g (NH 4 ) 2 HP0 4 , stir until dissolved, and dil. 
to 50 mL with H 2 0. Add few drops thymol blue soln (0.1%: 
dissolve 0.1 g thymol blue in H 2 0, add enough IN NaOH 
to change color to blue, and dil. to 100 mL), and then add 
NH 4 OH until soln just turns yellow. Add 0.5 mL HC1 and 25 
mL 25% NH 4 OAc soln, and stir. Let stand at room temp, until 
ppt settles (ca 1 hr). Filter, and wash 10 times with hot 5% 
NH 4 NO^ soln. Ignite at 500-550° and weigh as FeP0 4 and 
A1P0 4 . 

Fuse ignited residue in Pt crucible with ca 4 g Na 2 C0 3 -K 2 C0 3 
(1 + 1) mixt. When fusion is complete, let crucible cool, add 
5 mL H 2 S0 4 , and heat until copious fumes of SO3 are evolved. 
Cool, transfer to flask, add H 2 0, and digest until soln is clear. 
Reduce Fe with Zn, cool, and titr. with 0.1 AT KMn0 4 . Correct 
for blank and calc. as % Fe or % Fe 2 3 . Calc. to FeP0 4 and 
subtract from total Fe- and A1P0 4 to obtain A1P0 4 . Correct 
for blank and report as A1 2 3 . 

Refs.: JAOAC 11, 203(1928); 16, 70(1933); 19, 70(1936). 

CAS-7784-30-7 (aluminum phosphate) 
CAS- 10045-86-0 (ferric phosphate) 



910.01 



Calcium in Plants 

Titrimetric Macro Method 

Final Action 



Transfer aliquot of Soln I, 920.08, to 200 mL beaker, add 
H 2 if necessary to vol. of 50 mL, heat to bp, and add 10 mL 
satd (NH 4 ) 2 C 2 4 soln and drop Me red (dissolve 1 g Me red 
in 200 mL alcohol). Almost neutze with NH 4 OH and boil until 
ppt is coarsely granular. Cool, add NH 4 OH (1+4) until color 
is faint pink (pH 5.0), and let stand 5=4 hr. Filter, and wash 
with H 2 at room temp, until filtrate is oxalate-free. (Reserve 
filtrate and washings for Mg detn, 920.09.) 

Break point of filter with Pt wire, and wash ppt into beaker 
in which Ca was pptd, using stream of hot H 2 0. Add ca 10 
mL H 2 S0 4 (1 +4), heat to ca 90°, add ca 50 mL hot H 2 0, and 
titr. with 0.05 N KMn0 4 . Finally add filter paper to soln and 
complete titrn. 

Refs.: J. Biol. Chem. 7, 83(1910). JAOAC 4, 392(1921); 16, 
70(1933). 

CAS-7440-70-2 (calcium) 



921.01 



Calcium in Plants 

Titrimetric Micro Method 

Final Action 



Weigh 2 g sample into small crucible and ignite in furnace 
at 500-550°. Dissolve ash in HCl (1+4) and transfer to 100 
mL beaker. Add 5 mL HCl and evap. to dryness on steam 
bath to dehydrate Si0 2 . Moisten residue with 5 mL HCl, add 
ca 50 mL H 2 0, heat few min on steam bath, transfer to 100 
mL vol. flask, cool quickly to room temp., dil. to vol., shake, 
and filter, discarding first portion of filtrate. 



Pipet 15 mL aliquot into conical-tip centrf. tube contg 2 mL 
satd (NH 4 ) 2 C 2 4 soln and 2 drops Me red (dissolve 1 g Me 
red in 200 mL alcohol). Add 2 mL HOAc (1+4), rotating tube 
to mix contents thoroly. Add NH 4 OH (1+4), while intermit- 
tently rotating tube, until soln is faintly alk.; then add few 
drops of the HOAc until color is faint pink (pH 5.0). (It is 
important at this point to rotate tube so that last bit of liq. in 
conical tip has required color.) Let stand >4 hr; then centrf. 
15 min. (Ppt should be in firm lump in tip of tube.) Remove 
supernate, using suction device, Fig. 921.01, taking care not 
to disturb ppt. Wash ppt by adding 2 mL NH 4 OH (1+49), 
rotating tube to break up ppt. (It may be necessary to jar tube 
sharply.) Centrf. 10 min, again remove supernate, and wash 
with reagent as before. Repeat washing of ppt 3 times. 

After removing last supernate, add 2 mL H 2 S0 4 (1+4) to 
tube, break up ppt as before, heat on steam bath to 80-90°, 
and titr. in tube with 0.02N KMn0 4 , rotating liq. during titrn 
to attain proper end point. If tube cools to <60° during titrn, 
as indicated by slow reduction of KMn0 4 , reheat in steam bath 
few min and complete titrn. Perform blank on identical vol. 
H 2 S0 4 in similar tube heated to same temp, to det. vol. KMn0 4 
soln necessary to give end point color. Subtract this value from 
buret reading. 1 mL 0.02N KMn0 4 - 0.000400 g Ca. Report 
as % Ca. 

Refs.: J. Biol. Chem. 47, 475(1921); 50, 527, 537(1922). 
JAOAC 14, 216(1931); 16, 71(1933); 19, 71(1936). 

CAS-7440-70-2 (calcium) 



951.01 



Cobalt in Plants 

Nitrosocresol Method 

Final Action 1965 



(Caution: See safety notes on distillation, toxic solvents, car- 
bon tetrachloride, and nitroaromatics.) 

A. Reagents 

(Make all distns in Pyrex stills with J joints. Store reagents in 
g-s Pyrex bottles.) 

(a) Redistilled water. — Distil twice, or pass thru column of 
ion exchange resin (IR-IOOA, H-form, or equiv.) to remove 
heavy metals. 




FIG. 921.01 — Suction device used in micro method for deter- 
mining calcium 



44 



Plants 



AOAC Official Methods of Analysis (1990) 



(b) Hydrofluoric acid. — 48%. Procurement in vinyl plastic 
bottles is advantageous. 

(c) Perchloric acid. — 60%. No further purification neces- 
sary. 

(d) Hydrochloric acid. — (1 + 1). Add equal vol. HC1 to distd 
H 2 and distil. 

(e) Ammonium hydroxide. — (1 + I). Distil coned NH 4 OH 
into equal vol. redistd H 2 0. 

(f) Ammonium hydroxide. — 0.02N . Add 7 mL of the NH4OH 
(1 + 1) to 2.5 L redistd H 2 0. 

(g) Carbon tetrachloride. — Distil over CaO, passing dis- 
tillate thru dry, acid-washed filter paper. Used CC1 4 may be 
recovered as in 941.03A(a). 

(h) Dithizone. — Dissolve 0.5 g dithizone in 600-700 mL 
CCI4 (tech. grade is satisfactory). Filter into 5 L separator contg 
2.5-3.0 L 0.02 N NH 4 OH, shake well, and discard CC1 4 layer. 
Shake with 50 mL portions redistd CC1 4 until CC1 4 phase as 
it seps is pure green. Add 1 L redistd CC1 4 and acidify slightly 
with the HC1 (1 + 1). Shake the dithizone into CC1 4 layer and 
discard aq. layer. Store in cool, dark place, preferably in re- 
frigerator. 

(i) Ammonium citrate soln.— 40%. Dissolve 800 g citric acid 
in 600 mL distd H 2 0, and, while stirring, slowly add 900 mL 
NH 4 OH. Reaction is exothermic; take care to prevent spatter- 
ing. Adjust pH to 8.5, if necessary. Dil. to 2 L and ext with 
25 mL portions dithizone soln until aq. phase stays orange and 
CC1 4 remains predominantly green. Then ext soln with CC1 4 
until all orange is removed. 

(j) Hydrochloric acid.— AN. Dil. 16.6 mL of the HC1 
(1 + 1) to 1 L with redistd H 2 0. 

(k) Hydrochloric acid,— 0.0} N. Dil. 100 mL of the 0.1 JV 
HC1 to .1 L with redistd H 2 0. 

(1) Sodium hydroxide soln. — IN. Dissolve 40 g NaOH in 1 
L redistd H 2 0. 

(m) Borate buffer.— pH 7.8. Dissolve 20 g H 3 B0 3 in 1 L 
redistd H 2 0. Add 50 mL \N NaOH and adjust pH, if neces- 
sary. Equal vols borate buffer and 0.01 N HC1 should give soln 
ofpH7.9. 

(n) Borate buffer.— pH 9.1. To 1 L borate buffer, pH 7.8, 
add 120 mL IN NaOH and adjust pH, if necessary. 

(o) Skelly solve B . — Essentially «-hexane. Purify by adding 
20-30 g silica gel/L, let stand several days, and distil. Avail- 
able from Getty Refining and Marketing Co., PO Box 1650, 
Tulsa, OK 74102. 

(p) Cupric acetate soln.-— Dissolve 10 g Cu(OAc) 2 .H 2 in 
1 L redistd H 2 0. 

(q) o-Nitrosocresol soln. — Dissolve 8.4 g anhyd. CuCl 2 and 
8.4 g NH 2 OH.HCl in 900 mL H 2 0. Add 8 mL m-cresol (East- 
man Kodak Co., practical grade) and stir vigorously while 
slowly adding 24 mL 30% H 2 2 . Stir mech. 2 hr at room temp. 
(Standing for longer periods results in excessive decomposi- 
tion.) Add 25 mL HC1 and ext o-nitrosocresol with four 150 
mL portions Skellysolve B, (o), in large separator. Then add 
addnl 25 mL HC1 and again ext with four 150 mL portions 
Skellysolve B. Wash combined Skellysolve B exts twice with 
50-100 mL portions 0.17V HC1 and twice with 50-100 mL 
portions redistd H 2 0. Shake <9-nitrosocresol soln with succes- 
sive 50-100 mL portions 1% Cu(OAc) 2 soln until aq. phase 
is no longer deep blood-red. When light purple is evident, extn 
is complete. Discard Skellysolve B phase, acidify aq. soln of 
Cu salt with 25 mL HC1, and ext reagent with two 500 mL 
portions Skellysolve B; wash twice with 150-200 mL portions 
0. 17V HC1 and several times with 150-200 mL portions redistd 
H 2 0. Store c-nitrosocresol soln in refrigerator at ca 4°. Re- 
agent is stable >6 months. 

(r) Sodium o-nitrosocresol soln. — Ext 100 mL onitroso- 
cresol by shaking with two 50 mL portions borate buffer, pH 
9.1, in separator. (If this is carried out as 2 extns, resulting 



reagent is more coned. It is important that total vol. o-nitro- 
socresol soln equal total vol. buffer.) 

(s) Cobalt std solns. — (J) Stock soln. — Heat CoS0 4 .7H 2 
in oven at 250-300° to const wt (6-8 hr). Weigh exactly 0.263 
g of the C0SO4 and dissolve in 50 mL redistd H 2 and 1 mL 
H 2 S0 4 . Dil. to 1 L, (2) Working soln. — 0.5|xg/mL. Transfer 
5 mL stock soln to 1 L vol. flask and dil. to vol. with redistd 
H 2 0. 

(t) Hydroxylamine acetate buffer. — pH 5. 1 ±0.1. Dissolve 
10 g NH 2 OH.HCl and 9.5 g anhyd. NaOAc in 500 mL redistd 
H 2 0. 

B. Apparatus 

(a) Platinum dishes. — Approx. 70 mL; for ashing. 

(b) Automatic dispensing burets. — 100 mL; type that can 
be fitted to ordinary 5 lb reagent bottle and filled by means of 
aspirator bulb is most convenient. 

(c) Wooden separator rack. — Twelve-unit 125 mL sepa- 
rator size is convenient for dithizone extns. Rack is fitted across 
top with removable bar padded with sponge rubber so all 12 
separators can be shaken as unit. 

(d) Racks,— Consisting of 5 x 5 x 65 cm (2 x 2 x 25") 
wooden bars with holes drilled at close intervals to take 50 mL 
centrf. tubes fitted with No. 13 ¥ glass stoppers. To make these 
tubes, ream out necks of heavy- wall Pyrex centrf. tubes 
(Rockefeller Institute type) with t C rod and grind to take ¥ 
stopper. Place tubes upright in one section, and place other 
section (fitted with sponge rubber disks 13 mm thick in bottom 
of holes) across their tops. Fasten 2 sections at ends with re- 
movable rubber connectors made from ordinary tubing of con- 
venient size, so that any number of tubes can be shaken as 
unit. Use these tubes for reaction of Co with nitrosocresol, 
extn of complex into Skellysolve B, and washing of Skelly- 
solve B soln. 

(e) Shaking machine. — Mech. shaker giving longitudinal 
stroke of 5 cm at ca 180 strokes/min; use to make dithizone 
extns and to ext Co complex, or shake by hand. 

C. Cleaning of Glassware 

Clean 120 mL Pyrex separators for dithizone extns by ini- 
tially soaking 30 min in hot HN0 3 and rinsing several times 
with H 2 0. As added precaution, shake with several portions 
dithizone in CC1 4 . After use, clean by rinsing with H 2 0, drain, 
and stopper to avoid contamination. It is not necessary to clean 
every time with acid. Repeat HN0 3 cleaning if blanks are un- 
usually high. 

Clean 50 mL g-s Pyrex centrf. tubes by soaking 30 min in 
HNO3 followed by several rinsings in H 2 0. 

Completely submerge pipets in cylinder of chromic acid 
cleaning soln overnight, rinse several times with H 2 0, and sus- 
pend upright in rack to dry. 

Wash all other glassware thoroly in detergent and rinse well 
with tap H 2 followed by dip in chromic acid cleaning soln. 
Rinse off cleaning soln with tap H 2 followed by several distd 
H 2 rinses. 

Clean Pt by scrubbing with sea sand followed by boiling in 
HO (1+2) 30 min, and rinse several times with H 2 0. 

D. Preparation of Sample 

See 922.02(a). Oven-dry all plant material 48 hr and prep, 
for ashing by either of following methods: 

(a) Grind material in Wiley mill equipped with stainless steel 
sieve, mix thoroly by rolling, and sample by quartering. 

(b) Using stainless steel shears, cut material by hand fine 
enough for convenient sampling. 

E. Ashing of Samples 

(Caution: See safety notes on distillation, hydrofluoric acid, 
and perchloric acid.) 



AOAC Official Methods of Analysis (1990) 



Metals 



45 



Weigh 6 g dry plant tissue into clean Pt dish. Cover with 
Pyrex watch glass and place in cool furnace; heat slowly to 
500° and hold at this temp, overnight. Remove sample and 
cool. Wet down ash carefully with fine stream redistd H 2 0. 
From dispensing buret, slowly add 2-5 mL HC10 4 , dropwise 
at first to prevent spattering. Add ca 5 mL HF, evap. on steam 
bath, transfer to sand bath, and keep at medium heat until fum- 
ing ceases. 

Cover with Pyrex watch glass, return to partially cooled fur- 
nace, heat gradually to 600°, and keep at this temp. 1 hr. Re- 
move sample and cool. Add 5 mL HC1 (1 + 1) and ca 10 mL 
redistd H 2 0. Replace cover glass and warm on steam bath to 
dissolve. (Usually clear soln essentially free of insol. material 
is obtained.) Transfer sample to 50 mL vol. flask, washing 
dish several times with redistd H 2 0, dil. to vol., and mix tho- 
roly. (Pt dishes can ordinarily be used several times between 
sand and acid cleanings.) 

F. Dithizone Extraction 

(Caution: See safety notes on distillation, perchloric acid, and 
carbon tetrachloride.) 

Transfer suitable aliquot (2-3 g dry material) to 120 mL 
separator (use petroleum jelly as stopcock lubricant). Add 5 
mL NH 4 citrate soln and 1 drop phthln; adjust to pH 8.5 with 
NH 4 OH (1 + 1). If ppt forms, add addnl NH 4 citrate. Add 10 
mL dithizone in CC1 4 and shake 5 min. Drain CC1 4 phase into 
100 mL beaker. Repeat as many times as necessary, using 5 
mL dithizone soln and shaking 5 min each time. Extn is com- 
plete when aq. phase remains orange and CC1 4 phase remains 
predominantly green. Then add 1.0 mL CC1 4 , shake 5 min, and 
combine with CC1 4 ext. Final 10 mL CC1 4 should be pure green. 
If not, extn was incomplete and must be repeated. 

Add 2 mL HC10 4 to combined CC1 4 exts, cover beaker with 
Pyrex watch glass, and digest on hot plate until colorless. Re- 
move cover glass and evap. slowly to dryness. (If sample is 
heated any length of time at high temp, when dry, losses of 
Co may occur. Heat only enough to evap. completely to dry- 
ness. If free acid remains, it interferes with next step where 
pH control is important.) 

Add 5 mL 0.0 IN HC1 to residue. Heat slightly to assure 
soln. If Cu is to be detd, transfer with redistd H 2 to 25 mL 
vol. flask, and dil. to vol. Transfer 20 mL aliquot to 50 mL 
g-s centrf . tube or 60 mL separator and reserve remainder for 
Cu detn, 953. 03B. If Cu is not to be detd, transfer entire acid 
soln with redistd H 2 to centrf. tube or separator. 

G. Determination 

Add 5 mL borate buffer, pH 7.8, and 2 mL freshly prepd 
Na o-nitrosocresol soln to sample soln. Add exactly 5 mL 
Skellysolve B and shake 10 min. Remove aq. phase by mod- 
erate suction thru finely-drawn glass tube. To Skellysolve B 
layer add 5 mL Cu(OAc) 2 soln and shake 1 min to remove 
excess reagent. Again remove and discard aq. phase. Wash 
Skellysolve B by shaking I min with 5 mL redistd H 2 0, re- 
moving aq. layer as before; finally shake Skellysolve B 1 min 
with 5 mL NH 2 OH -NaOAc buffer to reduce Fe. Transfer Skel- 
lysolve B soln of the Co complex to 5 cm cell and read in 
spectrophtr as close as possible to point of max. A, 360 nm. 

H. Blanks and Standards 

With each set of detns include ashing blank and Co stds of 
0.0, 0.5, and 1.0 |xg. Beer's law holds for this range. A of 
0.0 jULg point should be <0.05. If above, repurify o-nitroso- 
cresol by transferring alternately to aq. phase as Cu salt and 
to Skellysolve B phase as free compd after acidifying aq. phase. 

It is also advisable to include std sample with each set of 
samples to detect contamination or unusual losses of Co in 



method. Com. buckwheat flour contg 0.05 ppm Co has proved 
satisfactory for this purpose. 

/. Calculations 

Express results in terms of ppm Co, based upon dry wt of 

sample. 

ppm Co — (p,g Co/mL dithizone aliquot) 

X (mL total soln/g dry sample) 

Value for p,g Co is obtained from curve minus ashing blank. 

Refs.: JAOAC 34, 710(1951); 36, 405(1953). 

CAS-7440-48-4 (cobalt) 



953.02 



Cobalt in Plants 

Nitroso-R-Salt Method 

Final Action 1965 



A. Reagents 

Those listed in 951. 01 A, and following: 

(a) N itr o so -R- salt soln. — 0.2%. Dissolve 2 g powd nitroso- 
R-salt (Eastman Kodak Co., No. 1124) in redistd H 2 0, 
951.01A(a), and dil. to 1 L. 

(b) Dilute nitric acid. — (1 + 1). Dil. HNO3 with equal vol. 
H 2 and redistil in Pyrex app. Store in Pyrex bottles. 

(c) Bromine water. — Satd soln of Br in redistd H 2 0, 
951.01A(a) 

(d) Citric acid soln. — 0.2 N. Use special reagent grade Pb- 
free citric acid. 

B. Preparation and Ashing of Samples 

Proceed as in nitrosocresol method, 951.010 and E, thru 
"(Usually clear soln essentially free of insol. material is ob- 
tained.)" except use 10 g instead of 6 g dry plant tissue. 

C. Dithizone Extraction 

Transfer entire soln to 120 mL separator, and proceed as in 
951. 01F, thru "If free acid remains . . . pH control is impor- 
tant.)" Dissolve in 1 mL citric acid soln, (d), transfer to 25 
mL vol. flask, and dil. to vol. with redistd H 2 0, 951.01A(a). 

D. Determination 

Transfer suitable aliquot (ca 8 g dry material) of citric acid 
soln, 953.02C, to 50 mL beaker. Evap. to 1-2 mL. Add 3 
mL borate buffer, 951.01A(n), and adjust pH to 8.0-8.5 with 
NaOH (check externally with phenol red). (Vol. <5 mL.) Add 
I mL nitroso-R-salt soln slowly with mixing. Boil 1-2 min and 
add 2 mL dil. HN0 3 . Boil 1-2 min, add 0.5-1.0 mL Br-H 2 0, 
cover with watch glass, and let stand warm 5 min. Boil 2-3 
min to remove excess Br (use effective fume removal device). 
Cool, and dil. to 10 or 25 mL (depending on length of light 
path in absorption cell). Transfer to cell and read at 500 nm 
within 1 hr. Prep, stds contg 0.5, 1, 2, 3, and 4 |xg Co and 
add 1 mL citric acid soln, 352.03A(d), to each. Proceed as 
for unknowns, beginning "Evap. to 1-2 mL." 

Ref.: JAOAC 36, 405(1953). 

CAS-7440-48-4 (cobalt) 



953.03 



Copper in Plants 

Colorimetric Method 

Final Action 1965 



A. Reagents 

Those listed in 951.01 A, and following: 
(a) Sodium diethyldithiocarbamate soln.- 

prepd in redistd H 2 0, 951.01A(a). 



-0.1%. Freshly 



46 



Plants 



AOAC Official Methods of Analysis (1990) 



(b) Copper std soln. — I |mg/mL. Dissolve 0.3929 g 
CuS0 4 .5H 2 in redistd H 2 0, 951.01A(a), add 5 mL H 2 S0 4 , 
dil. to 1 L, and mix. Take 10 mL aliquot, add 5 mL H 2 S0 4 , 
dil to 1 L, and mix. 

B. Determination 

Transfer aliquot (0.5-1 g dry material) from soln obtained 
from 951.01F or 953. 02C to 125 mL separator. Add 2 mL 
NH 4 citrate soln, 1 drop phthln, 5 mL Na diethyldithiocar- 
bamate soln, and NH 4 OH (1 + 1), 951.01A(e), until pink. Add 
10 mL CC1 4 and shake 5 min. Drain CC1 4 , centrf. 5 min, trans- 
fer to absorption cell, and read at max. A, ca 430 nm. 

Prep, std curve with 0, 1, 5, 10, 15, and 20 |ULg Cu treated 
as above. 

Ref.: JAOAC 36, 405(1953). 

CAS-7440-50-8 (copper) 



937.03 



Iron in Plants 

Colorimetric Method 

Final Action 



120 g HO Ac to 1 L with H 2 0. 
— 1%. Dissolve 1 g NH 4 citrate 



A Reagents 

(a) Acetic acid. — 2M. Oil. . 

(b) Ammonium citrate soln.- 
in H 2 and dil. to 100 mL. 

(c) Bromophenol blue indicator. — 0.04%. Grind 0.1 g bro- 
mophenol blue in mortar with 3 mL 0.05W NaOH, transfer to 
vol. flask, and dil. to 250 mL with H 2 0. 

(d) Buffer solns.-(l) pH 3.5.~~~Mix 6.4 mL 2M NaOAc 
with 93.6 mL 2Af HO Ac and dil. to 1 L. (2) pH 4. 5. —Mix 
43 mL 2M NaOAc with 57 mL 2M HOAc and dil. to 1 L. 

(e) Hydroquinone soln. — Dissolve 1 g hydroquinone in 100 
mL pH 4.5 buffer, (d)(2). Keep in refrigerator, and discard 
when any color develops. 

(f) o-Phenanthroline soln. — Dissolve 1 g o-phenanthro- 
line.H 2 in H 2 0, warming if necessary, and dil. to 400 mL. 

(g) Sodium acetate soln. — 2M. Dissolve 272 g NaOAc.3H 2 
in H 2 and dil. to 1 L. 

(h) Iron std soln. — 1 mg/mL. Dissolve 1 g electrolytic Fe 
in 50 mL H 2 S0 4 (1+9), warming if necessary to hasten re- 
action. Cool, and dil. to 1 L with H 2 0. 

B. Preparation of Sample 

(Caution: See safety notes on distillation, hydrofluoric acid, 
and sulfuric acid.) 

Use Soln I, 920.08, or if Soln I is not available, weigh sam- 
ples of finely ground plant material (1-5 g, depending on Fe 
content) into porcelain crucibles with smooth inner surfaces, 
and ash overnight at 500-550° in furnace. Cool, add 5 mL 
HC1 (1 + 1), and heat on steam bath 15 min to dissolve Fe and 
to hydrolyze pyrophosphate. Filter into 100 mL vol. flask. 
Transfer insoJ. residue to filter and wash 5 times with 3 mL 
portions hot HO (1 + 100), then with hot H 2 until washings 
are Cl-free. Ignite paper and any remaining C in Fe-free Pt 
crucible. Cool, add 2 drops H 2 S0 4 and 1 mL HF, and carefully 
evap. to S0 3 fumes. Cool, add few drops HC1 (1+1), and 
warm. Filter and wash as before into same vol. flask, dil. to 
vol., and mix. 

C. Determination 

Pipet identical aliquots of Soln 1, 920.08, or sample soln, 
937. 03B, into 25 mL vol. flask and into test tube or small 
erlenmeyer. Add 5 drops bromophenol blue indicator to ali- 
quot in test tube, and titr. with 2M NaOAc soln until color 



matches that of equal vol. of pH 3.5 buffer contg same amt 
of indicator. Add 1 mL hydroquinone soln and 2 mL o-phen- 
anthroline soln to aliquot in vol. flask, and adjust pH to 3.5 
by adding same vol. NaOAc soln found necessary for aliquot 
in test tube. If turbidity develops upon adjusting pH of aliquot 
in test tube, add I mL NH 4 citrate soln to vol. flask before 
adding the NaOAc soln. Dil. to vol., mix, and let stand .1 hr 
for complete color development, and measure A at max., ca 
510 nm. 

Prep, curve relating A to mg Fe in 25 mL by treating series 
of solns contg amts of Fe that cover usable range of instrument 
exactly as described for unknowns, detg their respective read- 
ings at max. A, ca 510 nm, and plotting these against corre- 
sponding concns of Fe. H 2 may be used as ref., and blanks 
detd to correct for amt Fe in reagents used, or blank soln itself 
may be made basis of comparison. 

Refs.: Ind. Eng. Chem. Anal. Ed. 9, 67(1937); 10, 13(1938). 
JAOAC 25, 555(1942); 27, 526(1944). 

CAS-7439-89-6 (iron) 



936.02 



Iron in Plants 

Titrimetric Method 
Final Action 



Take appropriate aliquot of Soln I or of soln prepd as in 
937.03B, and oxidize Fe by adding soln of KMn0 4 (1 + 1000) 
dropwise until very faint permanganate color persists. Add 5 
mL 10% NH 4 SCN and titr. with dil. TiCl 3 soln until red color 
disappears. (To prep, appropriate TiCl 3 soln, boil 5-10 mL 
20% TiCl 3 with 50 mL HC1 few min, cool, and dil. to 1 L. 
Stdze against std Fe soln, keep in dark in well-filled container, 
and restdze each time it is used, or every few hr when many 
detns are being made. Discard when decomposition is indi- 
cated by loss of color and increased titer against std.) 

Refs.: JAOAC 19, 359(1936); 27, 526(1944). 

CAS-7439-89-6 (iron) 



920.09* Magnesium in Plants 

Gravimetric Method 

Final Action 
Surplus 1989 



See 3.039, 14th ed. 



921.02 



Manganese in Plants 

Colorimetric Method 

Final Action 



To aliquot of Soln I, 920.08, contg 0.2-0.5 g ash, add 15 
mL H 2 S0 4 and evap. to ca 30 mL. Add 5-10 mL HN0 3 and 
continue evapn. (Do not evap. until dense fumes appear, be- 
cause Fe 2 (S0 4 ) 3 then dissolves with difficulty. HN0 3 may be 
present, but not HC1.) Add H 2 0, little at time, heat until Fe 
salts dissolve, and dil. to ca 150 mL. Add 0.3 g KlG 4j or its 
equiv. in HI0 4 , in small portions, boil few min or until color 
of KM11O4 shows no further increase in intensity, and let cool. 

Prep, std as follows: To vol. H 2 equal to sample add 15 
mL H 2 S0 4 and enough pure Fe(N0 3 ) 3 , free from Mn, to equal 
approx. amt of Fe in sample. Add measured vol. 0.1 N KMn0 4 
until color is slightly darker than sample, then add 0.3 g K10 4 , 
and boil few min. When cool, transfer sample and std to 250 



AOAC Official Methods of Analysis (1990) 



Metals 



47 



mL vol. flasks and dil. to vol. with H 2 0. (If color is weak, it 
may be necessary to dil. to <250 mL.) Measure A with pho- 
tometer or spectrophtr set at max., ca 530 nm. Report as % 

Mn. 

Ref.: JAOAC 4, 393(1921). 
CAS-7439-96-5 (manganese) 



960.05 Molybdenum in Plants 

Colorimetric Method 

First Action 1960 
Final Action 1965 

A. Apparatus 

Photoelectric colorimeter or spectrophotometer . — Capable 
of isolating band at ca 465 nm. (Photometer equipped with 
filter with max. T at 440-460 nm and J cm cells of 10 mL 
capacity is suitable.) 

0. Reagents 

(a) Isoamyl alcohol. — Reagent grade 3-methyI-l-butanol, 
bp 128-132°. 

(b) Dilute hydrochloric acid. — (7) 20% soln. — Dil. coned 
HCl to ca 20% HCl (1 + 1 .85). (2) 6N soln.—- Stdze to second 
decimal place. 

(c) Iron std soln. — 100 (xg/mL. Dissolve 0.7022 g 
Fe(NH4) 2 (S0 4 > 2 .6H 2 in H 2 0, add 1 mL H 2 S0 4 , and dil. to 
1 L. 

(d ) Molybden am std solus . — (1 ) Stock soln . — 1 00 jxg /mL . 
Dissolve 0.0920 g (NH 4 ) 6 Mo 7 24 .4H 2 in H 2 and dil. to 500 
mL. (2) Working soln. — 5 fxg/mL. Dil. 25 mL stock soln to 
500 mL. 

(e) Potassium thiocyanate soln. — 20%. Dissolve 50 g KSCN 
in H 2 and dil. to 250 mL. 

(f) Sodium fluoride saturated soln. — Add 200 mL H 2 to 
ca 10 g NaF. Stir until satd and filter. 

(g) Stannous chloride solns. — (1) 20% soln. — Weigh 10 g 
SnCl 2 .2H 2 into beaker, add 10 mL 20% HCl, (b)(7), and 
heat until completely dissolved. Cool, add granule of metallic 
Sn, dil. to 50 mL with H 2 0, and store in g-s bottle. (2) 0.8% 
wash soln.— OH . 4 mL 20% soln to 100 mL with H 2 0. 

C. Determination 

{Caution: See safety notes on wet oxidation, nitric acid, and 
perchloric acid.) 

Weigh 1-5 g finely ground sample, contg <35 u-g Mo, into 
200 mL tall-form Pyrex beaker. To 1, 2, or 5 g samples add 
10, 15, or 35 mL HN0 3 , resp. Include 2 beakers for blanks. 
Cover beaker with cover glass, and let stand ca 15 min; then 
heat cautiously on steam bath or hot plate at ca 100°, avoiding 
frothing over top. If froth approaches cover glass, remove beaker 
from heat until frothing subsides; then continue heating. Di- 
gest, usually ca 2 hr, until most of solids disappear. 

Cool to room temp. If contents should go to dryness, add 
few mL HN0 3 . Add 6 mL 70-72% HCIO4, cover beaker, place 
on hot plate, and gradually raise temp, so that contents boil 
vigorously but do not bump. Continue heating until digestion 
is complete as indicated by liq. becoming colorless or pale 
yellow. If necessary, make repeated addns of HN0 3 and HCIO4 
and continue to digest until C is completely oxidized. 

After digestion is complete, place cover glass slightly to one 
side of top of beaker, or replace it with elevated watch glass, 
and evap. just to dryness or until residue appears only slightly 
moist. Remove beaker from hot plate, and cool. Wash down 
sides of beaker and underside of cover glass with few mL H 2 0, 



return to hot plate, and boil few min. Remove from hot plate, 
cool, and again rinse sides of beaker and cover glass with small 
amt H 2 0. 

Add 2 drops Me orange and neutze with NH 4 OH. Add 67V 
HCl, dropwise with stirring, until soln is just acid; then add 
8.2 mL excess to give final concn of ca 3% HCl. Add 2 mL 
satd NaF soln, and 1 mL Fe soln, if sample contains <100 
jxg Fe. 

Transfer soln to 125 mL separator and dil. to 50 mL with 
H 2 0. Add 4 mL 20% KSCN soln, mix thoroly, and add 1.5 
mL 20% SnCl 2 soln. Mix again, and from buret or pipet, add 
exactly 15 mL isoamyl alcohol. Stopper separator and shake 
vigorously 1 min, let phases sep., and drain and discard aq. 
layer. Ext into alcohol without delay, since colored complex 
is somewhat unstable in aq. soln. 

Add 25 mL freshly prepd 0.8% SnCl 2 wash soln, and shake 
gently 15 sec. Let phases sep., and drain and discard aq. layer. 
Transfer isoamyl alcohol soln to centrf. tube, and centrf. 5 min 
at ca 2000 rpm to remove H 2 droplets. If alcohol layer is not 
clear, recentrf. Stopper tubes to prevent evapn, if A readings 
cannot be made immediately. 

Compare unknown solns with isoamyl alcohol at ca 465 nm 
in spectrophtr, and make appropriate corrections in A readings 
for Mo in blanks. Obtain Mo concn from calibration curve 
relating A readings to conens of series of solns of known Mo 
content. 

Prep, calibration curve for instrument used, as follows: Place 
aliquots of working std soln contg 0, 5, 10, 15, 20, 25, 30, 
and 35 |xg Mo, resp., into 200 mL tall-form beakers and carry 
them thru entire detn, beginning with digestion with HNO3 and 
HCIO4. Plot A against corresponding Mo conens. 

Refs.: JAOAC 36, 412(1956); 41, 309(1958); 43, 510(1960). 

CAS-7439-98-7 (molybdenum) 



925.01* Potassium and Sodium in Plants 

Gravimetric Method 

Final Action 
Surplus 1974 



■See 3.015, 11th ed. 



956.01 Potassium and/or Sodium in Plants 
Flame Photometric Method 
Final Action 1965 

A. Reagents 

(a) Potassium stock soln. — 1000 ppm K. Dissolve L907 g 
dry KCl in H 2 and dil. to 1 L. 

(b) Sodium stock soln. — 1000 ppm Na. Dissolve 2.542 g 
dry NaCl in H 2 and dil. to 1 L. 

(c) Lithium stock soln. — 1000 ppm Li. Dissolve 6.108 g 
LiCl in H 2 and dil. to 1 L. (Needed only if internal std method 
of evaluation is to be used.) 

(d) Ammonium oxalate stock soln. — 0.24/V. Dissolve 17.0 
g (NH 4 ) 2 C 2 4 .H 2 in H 2 and dil. to 1 L. 

(e) Extracting solns . — (1 ) For potassium . — For internal std 
method, dil. required vol. LiCl stock soln to 1 L; otherwise 
use H 2 0. (2) For sodium. — To 250 mL NH 4 oxalate stock soln 
add required vol. LiCl stock soln (if internal std method is 
used) and dil. to 1 L. If internal std requirements are same for 
both Na and K detns, this reagent may be used as common 
extg soln. 



48 



Plants 



AOAC Official Methods of Analysis (1990) 



B. Preparation of Standard Solutions 

Dil. appropriate aliquots of stock solns to prep, series of stds 
contg K and/or Na in stepped amts (including 0) to cover in- 
strument range, and Li and NH 4 oxalate (if required) in same 
concns as in corresponding extg solhs. (If common extg soln 
is used, 1 set of stds contg both K and Na suffices.) 

C. Sample Extraction 

Transfer weighed portion of finely ground and well mixed 
sample to erlenmeyer of at least twice capacity of vol. of extg 
soln to be used. Add measured vol. extg soln, stopper flask, 
and shake vigorously at frequent intervals during 2=15 min. 
Filter thru dry, fast paper. If paper clogs, pour contents onto 
addnl fresh paper and combine filtrates. Use filtrate for detn. 

Note: Do not make exts more coned than required for in- 
strument because there is tendency toward incomplete extn as 
ratio of sample wt to vol. extg soln increases. Prep. sep. exts 
for K and Na when their concns in sample differ greatly. For 
K, use wt sample <0. 1 g/50 mL extg soln; for low Na concns 
use >1.0 g/50 mL extg soJn; and for higher concns, prep. 
weaker exts by reducing ratio of sample to extg soln rather 
than by dilg stronger exts. 

D. Determination 

(Caution: See safety notes on flame photometer.) 

Rinse all glassware used in Na detn with dil. HN0 3 , fol- 
lowed by several portions H 2 0. Protect solns from air-borne 
Na contamination. 

Operate instrument according to manufacturer's instruc- 
tions. Permit instrument to reach operating equilibrium before 
use. Aspirate portions of std solns toward end of warm-up pe- 
riod until reproducible readings for series are obtained. 

Run stds, covering concn range of samples involved, at fre- 
quent intervals within series of sample soln detns. Repeat this 
operation with both std and sample solns enough times to re- 
sult in reliable av. reading for each soln. Plot curves from 
readings of stds, and calc. % K and/or Na in samples. 

Ref.: J AOAC 39, 419(1956). 

CAS-7440-09-7 (potassium) 

CAS-7440-23-5 (sodium) 



935.03* Potassium in Plants 

Platinic Chloride Method 

Final Action 
Surplus 1974 



See 3.020, ilth ed. 



936.03* 



Potassium in Plants 

Perchloric Acid Method 

Surplus 1974 



See 3,021, 11th ed. 



935.04* Potassium in Plants 

Rapid Method for Potassium Only 
Surplus 1974 

See 3.022, llth ed. 



929.03* Sodium in Plants 

Uranyl Acetate Method 

Final Action 
Surplus 1989 

See 3.052-3.053, 14th ed. 



941.03 



A. Reagents 



Zinc in Plants 

Mixed Color Method 

Final Action 1965 



(Redistil all H 2 from Pyrex. Treat all glassware with HN0 3 

(1 + 1) or fresh chromic acid cleaning soln. Rinse repeatedly 

with ordinary distd H 2 and finally with Zn-free H 2 0.) 

(a) Carbon tetrachloride. — Use ACS grade without puri- 
fication. If tech. grade is used, dry with anhyd. CaC1 2 and 
redistil in presence of small amt CaO. (Used CC1 4 may be 
reclaimed by distn in presence of NaOH (1 + 100) contg small 
amts of Na 2 S 2 3 , followed by drying with anhyd. CaCI 2 and 
fractional distn in presence of small amts of CaO.) (Caution: 
See safety notes on distillation and carbon tetrachloride.) 

(b) Zinc std solns. — (I) Stock soln. — 1 mg/mL. Place 0.25 
g pure Zn in 250 mL vol. flask. Add ca 50 mL H 2 and 1 
mL H 2 S0 4 ; heat on steam bath until all Zn dissolves. Dil. to 
vol. and store in Pyrex vessel. (2) Working soln. — 10 p,g/mL. 
Dil. 10 mL stock soln to 1 L. Store in Pyrex vessel. 

(c) Ammonium hydroxide soln. — IN. With all-Pyrex app. 
distil NH 4 OH into H 2 0, stopping distn when half has distd. 
Dil. distillate to proper concn. Store in g-s Pyrex vessel. 

(d) Hydrochloric acid. — IN. Displace HC1 gas from HC1 
in glass flask by slowly adding equal vol. H 2 S0 4 from drop- 
ping funnel that extends below surface of the HC1. Conduct 
displaced HC1 gas thru delivery tube to surface of H 2 in re- 
ceiving flask (no heat is necessary). Dil. to proper concn. Use 
of 150 mL each of HC1 and H 2 S0 4 will yield 1 L purified HC1 
soln of concn >IN. 

(e) Diphenylthiocarbazone (dithizone) soln. — Dissolve 0.20 
g dithizone in 500 mL CC1 4 , and filter to remove insol. matter. 
Place soln in g-s bottle or large separator, add 2 L 0.027V NH 4 OH 
(40 mL IN NH 4 OH dild to 2 L), and shake to ext dithizone 
into aq. phase. Sep. phases, discard CC1 4 , and ext ammoniacal 
soln of dithizone with 100 mL portions CC1 4 until CC1 4 ext is 
pure green. Discard CC1 4 after each extn. Add 500 mL CC1 4 
and 45 mL IN HC1, and shake to ext dithizone into CC1 4 . Sep. 
phases and discard aq. phase. Dil. CC1 4 soln of dithizone to 
2 L with CC1 4 . Store in brown bottle in dark, cool place. 

(f) Ammonium citrate soln. — 0.5M. Dissolve 226 g 
(NH 4 ) 2 HQH 5 7 in 2 L H 2 0. Add NH 4 OH (80-85 mL) to pH 
of 8.5-8.7. Add excess dithizone soln (aq. phase is orange- 
yellow after phases have been shaken and sepd), and ext with 
100 mL portions CC1 4 until ext is full green. Add more dithi- 
zone if necessary. Sep. aq. phase from CC1 4 and store in Pyrex 
vessel. 

(g) Carbamate soln. — Dissolve 0.25 g Na diethyldithio- 
carbamate in H 2 and dil to 100 mL with H 2 0. Store in re- 
frigerator in Pyrex bottle. Prep, fresh after 2 weeks. 

(h) Dilute hydrochloric acid.— 0.02N '.Dil. lOOmLlJVHCl 
to5L. 

B. Preparation of Solutions 

To reduce measuring out reagents and minimize errors due 
to variations in composition, prep. 3 solns in appropriate amts 
from reagents and store in Pyrex vessels, taking care to avoid 



AOAC Official Methods of Analysis (1990) 



Metals 



49 



loss of NH 3 from Solns I and 2. Discard solns after 6-8 weeks 
because Zn increases slowly with storage. Det. std curve for 
each new set of reagents. Following amts of Solns 1 and 2 and 
2 L dithizone soln are enough for 100 detns: 

(1) Soln jf.— Dil.. 1 L 0.5M NH 4 citrate and 140 rnL \N 
NH 4 OH to 4 L. 

(2) Soln 2.— Dil. 1 L 0.5 M NH 4 citrate and 300 mL IN 
NH 4 OH to 4.5 L. Just before using, add 1 vol. carbamate soln 
to 9 vols NH3-NH4 citrate soln to obtain vol. of Soln 2 im- 
mediately required. 

Note: If Zn-free reagents have been prepd, they can be used 
to test chemicals for Zn. Certain lots of NH 4 OH and HC1 are 
sufficiently free of Zn to be used without purification. 

C. Ashing 

Ash 5 g finely ground, air-dried plant material in Pt dish in 
furnace at 500-550°. Include blank detn. Moisten ash with 
little H 2 0; then add 10 mL IN HCI (more if necessary) and 
heat on steam bath until all substances sol. in HCI are dis- 
solved. Add 5-10 mL hot H 2 0. Filter off insol. matter on 7 
cm paper (Whatman No. 42, or equiv., previously washed with 
two 5 mL portions hot IN HCI, then washed with hot H 2 
until HCl-free), and collect filtrate in 100 mL vol. flask. Wash 
filter with hot H 2 until washings are not acid to Me red. Add 
1 drop Me red (dissolve 1 g Me red in 200 mL alcohol), to 
filtrate in 100 mL flask; neutze with \N NH 4 OH and add 4 
mL 17V HCI. Cool, and dil. to vol. with H 2 0. 

D. First Extraction 

(Sepn of dithizone complex-forming metals from ash soln) 

Pipet aliquot of ash soln contg ^30 jxg Zn into 125 mL 
Squibb separator. Add 1 mL 0.2 N HCI for each 5 mL ash 
soln <10 mL taken, or 1 mL 0.27V NH 4 OH for each 5 mL 
>10 mL taken. (10 mL aliquot is usually satisfactory in anal- 
ysis of plant materials.) Add 40 mL Soln 1 and 10 mL dithi- 
zone reagent. Shake vigorously 30 sec to ext from aq. phase 
the Zn and other dithizone complex-forming metals that may 
be present; then let layers sep. At this point excess dithizone 
(indicated by orange or yellow-orange aq. phase) must be pres- 
ent. If excess dithizone is not present, add more reagent until, 
after shaking, excess is indicated. Shake down the drop of CC1 4 
ext from surface, and drain CC1 4 ext into second separator as 
completely as possible without letting any aq. layer enter stop- 
cock bore. Rinse down CC1 4 ext from surface of aq. layer with 
1-2 mL clear CCl 4 ; then drain this CC1 4 into second separator 
without letting aq. phase enter stopcock bore. Repeat rinsing 
process as often as necessary to flush ext completely into sec- 
ond separator. Add 5 mL clear CCl 4 to first separator, shake 
30 sec, and let layers sep. (CC1 4 layer at this point will appear 
clear green if metals that form dithizone complexes have been 
completely extd from aq. phase by previous extn.) Drain CC1 4 
layer into second separator and flush ext down from surface 
and out of separator as directed previously. If last ext does not 
possess distinct clear color, repeat extn with 5 mL clear CC1 4 
and flushing-out process until complete extn of dithizone com- 
plex-forming metals is assured; then discard aq. phase. 

E. Second Extraction 

(Sepn of Cu by extn of Zn into 0.02 W HCI) 

Pipet 50 mL 0.02N HCI into separator contg CC1 4 soln of 
metal dithizonates. Shake vigorously 1.5 min, and let layers 
sep. Shake down drop from surface of aq. phase, and as com- 
pletely as possible drain CC1 4 phase contg all Cu as dithizon- 
ate, without letting any aq. phase, which contains all the Zn, 
enter stopcock bore. Rinse down CC1 4 ext from surface of aq. 



phase, and rinse out stopcock bore with 1-2 mL portions clear 
CCI4 (same as in first extn) until all traces of green dithizone 
have been washed out of separator. Shake down drop of CC1 4 
from surface of aq. phase, and drain CC1 4 as completely as 
possible without letting any aq. phase enter stopcock bore. Re- 
move stopper from separator and lay it across neck until small 
amt of CCI4 on surface of aq. phase evaps. 

F. Final Extraction 

(Extn of Zn in presence of carbamate reagent) 

Pipet 50 mL Soln 2 and 10 mL dithizone soln into 50 mL 
0.027V HCI soln contg the Zn. Shake 1 min and let phases sep. 
Flush out stopcock and stem of separator with ca 1 mL CC1 4 
ext; then collect remainder in test tube. Pipet 5 mL ext into 
25 mL vol. flask, dil. to vol. with clear CC1 4 , and measure A 
with spectrophtr set at absorption max., ca 525 nm. (Caution: 
Protect final ext from sunlight as much as possible and read 
within 2 hr.) 

Det. Zn present in aliquot from curve relating A and concn, 
correct for Zn in blank, and calc. % Zn in sample. 

G. Standard Curve 

Place 0, 5, 10, 15, 20, 25, 30, and 35 mL Zn working std 
soln in 100 mL vol. flasks. To each flask add 1 drop Me red 
and neutze with 1 N NH 4 OH; then add 4 mL 1 N HCI and dil. 
to vol. Proceed exactly as for ash solns, beginning with first 
extn, and using 10 mL aliquots of each of the Zn solns (0, 5, 
10, 15, 20, 25, 30, and 35 fxg Zn, resp.). Construct std curve 
by plotting |xg Zn against A. 

Refs.: Ind. Eng. Chem. Anal. Ed. 13, 145(1941). JAOAC 
24, 520(1941). 

CAS-7440-66-6 (zinc) 



953.04 



Zinc in Plants 

Single Color Method 

Final Action 1965 



A. Reagents 

See 941. 03A and B plus following: 



vol. dithizone soln, 



(a) Dilute dithizone soln. — Dil. 
941.03A(e), with 4 vols CC1 4 . 

(b) Carbamate soln. — Dissolve 1.25 g Na diethyldithio- 
carbamate in H 2 and dil. to 1 L. Store in refrigerator and 
prep, fresh after long periods of storage. 

(c) Dilute ammonium hydroxide. — Dil. 20 mL IN NH 4 OH, 
941.03A(c), to 2 L. 

B. Ashing 

Weigh 2 g sample finely ground plant material into well- 
glazed porcelain, Vycor, or Pt crucible, include crucible for 
blank detn, and heat in furnace at 500-550° until ashing is 
complete. Cool, moisten ash with little H 2 0, add 10 mL \N 
HCI (more if necessary to ensure excess of acid), and heat on 
steam bath until all sol. material dissolves. Add few mL hot 
H 2 and filter thru quant, paper into 200 mL vol. flask. Wash 
paper with hot H 2 until washings are not acid to Me red. 
Add 2 drops Me red soln to filtrate, neutze with \N NH 4 OH, 
add exactly 3.2 mL IN HCI, dil. to vol. with H 2 0, and mix. 

C. Formation of Zinc Dithizonate 

(Removal of interferences and sepn of excess dithizone) 

Pipet aliquot of ash soln contg ^15 jxg Zn into 125 mL 
amber glass separator. (25 mL aliquot is usually satisfactory.) 
If necessary to use different vol., add 0.4 mL 0.2N HCI for 



50 



Plants 



AOAC Official Methods of Analysis (1990) 



each 5 mL less, or 0.4 mL 0.2 TV NH 4 OH for each 5 mL more, 
than 25 mL taken. If <25 mL of the soln is taken, add H 2 
to 25 rnL. 

Add 10 mL dithizone reagent, 941.03A(e), to aliquot in sep- 
arator and shake vigorously 1 min. Let layers sep. and discard 
CC1 4 layer. Add 2 mL CC1 4 to aq. so]n, let layers sep., and 
discard CC1 4 . Repeat this rinsing once. Then add 5 mL CC1 4 , 
shake vigorously 15 sec, let layers sep., and discard CC1 4 . 
Rinse once more with 2 mL CC1 4 as above. Discard CC1 4 layer 
and let CC1 4 remaining on surface of soln in funnel evap. be- 
fore proceeding. 

Add 40 mL NH 4 citrate Soln 1 , 941.03B(1), 5 mL carbamate 
soln, 954.04A(b), and 25 mL dil. dithizone reagent, 
954.04A(a). Accurately add carbamate and dithizone reagents 
from pipet or buret. Shake vigorously 1 min. Let layers sep. 
and draw off aq. layer thru fine tip glass tube connected to 
aspirator with rubber tubing. To remove excess dithizone from 
CC1 4 layer, add 50 mL 0.017V NH 4 OH and shake vigorously 
30 sec. 

D. Determination 

Dry funnel stem with pipestem cleaner and flush out with 
ca 2 mL of the Zn dithizonate soln. Collect adequate portion 
of remaining soln in 25 mL erlenmeyer, or other suitable con- 
tainer, and stopper tightly. (Amber glass containers are con- 
venient, but colorless glassware will suffice if solns are kept 
in dark until A readings are made.) 

Measure A of each soln against CC1 4 with spectrophtr set at 
absorption max., ca 535 nm. Correct for Zn in blank detns. 
Calc. amt Zn present in soln from curve relating concn and A. 

E. Standard Curve 

Into 200 mL vol. flasks place 0, 2, 4, 6, 8, 10, 12, and 14 
mL, resp., Zn working std soln. To each flask add 2 drops 
Me red soln, neutze with 17V NH 4 OH, add 3.2 mL 17V HC1, 
and dil. to vol. with H 2 0. Pipet 25 mL aliquots of each of 
these solns, contg 0, 2.5, 5, 7.5, 10, 12.5, 15, and 17.5 jxg 
Zn, resp., into amber glass separators, and proceed as for ash 
solns, 954. 04C, beginning with second par. Det. A of each 
soln and plot values against corresponding amts Zn. 

Ref.: JAOAC 36, 397(1953). 

CAS-7440-66-6 (zinc) 



NONMETALS 



930.06 



Arsenic in Plants 

Titrimetric Method 

Final Action 



Prep, soln as in 963. 21 C. Proceed as in 963. 2 ID, or take 
aliquot and det. as in 925.02, beginning ". . . add 3 mL H 2 S0 4 

CAS-7440-38-2 (arsenic) 



958.03 Boron in Plants 

Quinalizarin Method 

First Action 1958 
Final Action 1965 

A. Reagents 

(a) Dilute sulfuric acid. — 0.367V. Dil. 10 mL H 2 S0 4 to 
1 L. 



(b) Calcium hydroxide saturated soln. — Filter before use. 

(c) Quinalizarin soln.- — Dissolve 45 mg quinalizarin in 1 L 
95-96% H 2 S0 4 . 

(d) Boron std soln. — 0.5 mg B/mL. Dissolve 2.860 g H 3 B0 3 
and dil. to 1 L with H 2 0. Prep, working stds by further diln 
with H 2 0. 

B. Determination 

Place 1.00-2.00 g dry, ground plant material in Pt or Si0 2 
dish. Add 5 mL satd Ca(OH) 2 soln and dry at 105°. Carefully 
volatilize over burner, ash in furnace 1 hr at 600°, and cool. 
Add exactly 10 or 15 mL 0.36 A' H 2 S0 4 , break up ash with 
glass rod, stir gently, and filter. Transfer 2 mL filtrate to col- 
orimeter tube, add an exact amt (e.g., 15 mL) quinalizarin 
reagent, stopper, and mix by swirling gently. Let stand at room 
temp. 24 hr (or until both unknowns and stds have cooled to 
same temp.). Shake tube again immediately before reading in 
photoelec. colorimeter (620 nm filter). 

Adjust colorimeter to 100% T with blank soln prepd as above 
but using 2 mL H 2 in place of sample soln. Prep, std curve 
with series of stds contg 0.5-10 fxg B/mL. 

Ref.: JAOAC 41, 304(1958). 

CAS-7440-42-8 (boron) 



928.04* 



Chloride in Plants 

Gravimetric Method 

Final Action 
Surplus 1989 



See 3.069-3.070, 14th ed. 



915.01 



Chloride in Plants 

Volumetric Method I 

Final Action 



(Since precision of this titrn is considered to be ±0.2 mg CI, 
accuracy of 1.0% requires samples contg >20 mg.) 

A. Reagents 

(a) Silver nitrate std soln. — 1 mL = 0.00355 g CI. Prep, 
soln slightly stronger than 0.1 TV, stdze as in 941. 18E, and ad- 
just to exactly 0. IN. 

(b) Ammonium or potassium thiocyanate std soln. — 0.17V. 
Prep, soln slightly stronger than 0.17V, stdze as in 941.18D(b), 
and adjust to exactly 0.1 TV. 

(c) Ferric indicator. — Satd soln of FeNH4(S0 4 )2.12H 2 0. 

(d) Nitric acid. — Free from lower oxides of N by dilg HNO3 
with ca V4 vol. H 2 0, and boiling until perfectly colorless. 

B. Determination 

To prepd soln, 928. 04A, add known vol. std AgN0 3 soln 
in slight excess. Stir well, filter, and wash AgCl ppt thoroly. 
To combined filtrate and washings add 5 mL ferric indicator 
and few mL HNO3, and titr. excess Ag with thiocyanate std 
soln to permanent light brown. From mL AgN0 3 used, calc. 
%C1. 

Refs.: Sutton, "Systematic Handbook of Volumetric Analy- 
sis," 11th ed., 1924, p. 146. J. Am. Chem. Soc. 37, 
1128(1915). 

CAS-7782-50-5 (chlorine) 



AOAC Official Methods of Analysis (1 990) 



NONMETALS 



51 



935.05 



Chloride in Plants 

Volumetric Method II 

Final Action 



A. Reagents 

(a) Potassium iodide std soln. — I mL - 1 mg CI. Weigh 
4.6824 g pure (ACS) KI, dried to const wt at 105-150°, dis- 
solve in H 2 0, and dil. to 1 L. 

(b) Silver nitrate stock soln. — Approx. 0.37V. 1 mL = ca 
10 mg CI. Dissolve 48 g AgNO, in H.O, filter, and dil. to 
1 L. 

(c) Silver nitrate std soln. — Dil. 100 mL reagent (b) to ca 
900 mL and adjust by stdzg against reagent (a) so that 1 mL 
= 1 mg CI. 

(d) Chloride-free starch indicator. — For each 100 mL final 
soln take 2.5 g sol. starch and make to paste with cold H 2 0. 
Stir out lumps, add 25-50 mL more cold H 2 0, and stir or 
shake 5 min. Centrf. , decant, and discard liq. Repeat extn 3 
times and finally transfer residue to flask contg proper amt of 
boiling H 2 0. Stir again, heat to bp, cover with small beaker, 
and cool under tap, shaking occasionally. 

(e) Dilute sulfuric acid. — Add 35 mL H 2 S0 4 to each 1 L 
H 2 0, boil 5-10 min, and cool to room temp. 

(f) Iodine indicator. — To ca 20 g I in 500 mL g-s bottle 
add 400 mL dil. H 2 S0 4 , (e), and shake 10 min. Decant and 
discard first soln, since it may contain iodides. Repeat process 
and store soln in small g-s bottles. 

(g) Potassium permanganate soln. — Dissolve 60 g KMn0 4 
in 400 mL warm H 2 (ca 50°) and dil. to 1 L. 

(h) Potassium sulfate-copper sulfate mixture. — Thoroly mix 
16 parts K 2 S0 4 and 1 part CuS0 4 .5H 2 0. 

(i) Wash soln.— Mix 980 mL H 2 and 20 mL HN0 3 . 

B. Determination 

(Caution: See safety notes on wet oxidation, nitric acid, and 
permanganates.) 

Weigh sample contg 10-40 mg CI into beaker. (If >4 g is 
taken, use proportionately more HN0 3 and KMn0 4 soln.) Add 
10 mL 0.3iV AgN0 3 and stir until sample is thoroly soaked, 
adding little H 2 or warming if necessary. Add 25 mL HN0 3 , 
stir, add 5 mL KMn0 4 soln, and stir until frothing stops. Place 
mixt. in H 2 bath or on hot plate and keep just below bp. Stir, 
and wash down sides of beaker at intervals with min. FLO. 
After 20 min, or when reaction stops, add addnl KMn0 4 soln, 
little at time, until color begins to fade slowly. Dil. to ca 125 
mL with boiling H 2 and heat 10 min longer. (Beaker may 
stand in bath or on hot plate until ready to filter.) 

Filter while hot thru Whatman No. 5, or equiv. paper, with 
suction as follows: Place disk of 30-mesh stainless steel wire 
gauze or No. 40 filter cloth in bottom of 3" (7.6 cm) Hirsch 
funnel. Fold 9 cm paper over bottom of No. 11 rubber stopper, 
shaping it to funnel by making 9-10 folds up side of stopper. 
Place paper in funnel and apply strong suction. Wet paper and 
keep wet while fitting into funnel so as to avoid double thick- 
nesses of paper. Wash paper thoroly, first with H 2 and then 
with wash soln. Discard washings and rinse out flask. Decant 
thru filter and transfer ppt and sample residue to filter. If fil- 
trate is not turbid, or if it is only slightly opalescent, wash ppt 
thoroly, applying wash soln very gently, but keeping strong 
suction on filter. If combined filtrate and washings are clear, 
test for Ag. If turbid, reheat and pass thru filter, repeating until 
clear, and finally wash as above. If filtrate does not give def- 
inite test for Ag, repeat detn on smaller sample. 

Place paper and contents in Kjeldahl flask and add such amts 
of K 2 S0 4 -CuS0 4 mixt. and H 2 S0 4 as would be appropriate for 
protein detn on same kind and amt of sample, and digest sim- 



ilarly. (For 2 g grass, 8 g sulfate mixt. and 20 mL acid are 
enough.) When digest is cool, add 175 mL H 2 0, boil 5-10 
min, and cool to room temp. Titr. the Ag 2 S0 4 in Kjeldahl flask 
with KI std soln, using 5 mL starch indicator and 30 mL I 
indicator. (Add latter just before titrn.) Rinse neck of flask 
after each addn of KI when near end point and titr. until soln 
stays blue after shaking. If <30 mg CI is present, add starch 
and I so Ins at beginning. If larger but unknown amt is present, 
add 2 mL starch and 10 mL I indicator at beginning and titr. 
until end point approaches. Shake vigorously to coagulate ppt, 
add rest of starch and I solns, and proceed to end point. If 
known large amt is present, titr. to within 2 mL of end point, 
shake as above, add indicator reagents, and continue titrn. If 
end point is overrun, add 5 mL std AgN0 3 soln and titr. again. 
Blank detns are not necessary after testing reagents. If blank 
made by using pure sugar as sample is >0.05 mg, examine 
filter paper, distd H 2 0, and various reagents for CI. 

Refs.: JAOAC 18, 379(1935); 19, 72(1936). 

CAS-7782-50-5 (chlorine) 



975.04 Fluoride in Plants 

Potentiometric Method 

First Action 1975 
Final Action 1988 

(Rinse all plastic and glass containers with HCl (1+3) and H 2 

before use. Perform analyses in laboratory free from F; prep. 

samples in another laboratory.) 

A. Princ'tpie 

F is extd from dry, pulverized foliage with HN0 3 followed 
by aq. KOH. Slurry is adjusted to pH 5.5, and complexing 
agent and background F are added. Potential is measured with 
ion selective electrode and compared against calibration curve. 
Method is applicable to 10-2000 |ULg F/g dry wt leaf tissue 
not exposed to unusual amts of Al or other F-binding agents; 
it is not applicable to insol. inorg. F or F in org. combinations. 
Between-laboratory precision of individual analyses is ±20% 
at 30 ppm F; ±10%, ^100 ppm F. Accuracy is 90-100%. 

B. Apparatus 

Electrometer. — Range ±200 mv with readability of 0. 1 mv 
(Model 701 or 701 A (replacement model SA720) digital pH/ 
mv meter, Orion Research Inc., or equiv.) or expanded scale 
pH meter with mv mode of operation, with F ion selective 
electrode (No. 94-09 single electrode, Orion Research Inc., 
or equiv.) and reference electrode (No. 90-01 single junction, 
Orion Research Inc., or equiv.). Check system at intervals to 
assure adherence to following performance criteria: Using 
technic of 975.04D, system should reach equilibrium (A E <0.2 
mv/min) within 5 min with each F working std soln, checked 
in following sequence: 0.1, 0.2, 0.5, 2.0, and 10.0 ppm F. 
Replicate std solns should differ by ^1 mv. Calibration curve 
should be linear between 0.2 and 10.0 ppm and slope should 
be 57 ±2 mv per 10-fold change in F concn. If any parameter 
is not obtained, check electrodes, reagents, and electrometer. 
Maintain temp, control of ±1°. 

C. Reagents 

(Store all solns in tightly closed, plastic bottles.) 

(a) Nitric acid.—U) ION.— Add 63 mL HN0 3 to H 2 0, cool, 
and dil. to 100 mL. (2) 0.2N.— Dil. 5.0 mL 107V to 250 mL. 
(3) 0.05N.—DW. 5.0 mL 10/V to 1 L. 



52 



Plants 



AOAC Official Methods of Analysis (1990) 



(b) Potassium nitrate so In, — 0.4M. Dissolve 4.0 g KN0 3 
in H 2 anddiJ. to 100 mL. 

(c) Sodium citrate soln. — 0.8M. Dissolve 58.8 g Na ci- 
trate. 2H 2 in 200 mL H 2 0, adjust to pH 5.5 by drop wise addn 
of \0N HN0 3 , using pH meter, and dil to 250 mL with H 2 0. 

(d) Sodium citrate with fluoride soln. — 0AM citrate with 1 
ppm F. Dil. 125 mL 0.8M Na citrate soln and 25.0 mL 10 
ppm F std soln to 250 mL with H 2 0. 

(e) Fluoride std solns. — (/) Stock soln. — 100 ppm F. Dry 
ca 1 g NaF 2 hr at 110°. Accurately weigh 0.221 g NaF, dis- 
solve in H 2 0, and dil. to 1 L. (2) Intermediate soln, — 10 ppm 
F. Dil. 10.0 mL stock soln to 100 mL with H 2 0. (3) Working 
soln. — Prep, as in Table 975.04 in 100 mL vol, flasks. Prep. 
0.2 and 0.1 ppm solns fresh as needed. 

D. Preparation of Calibration Curve 

Place 25.0 mL 0.1 ppm F working std soln into plastic con- 
tainer contg stirring bar. Insert electrodes ca 12 mm into soln 
and stir mag. Record mv readings at 1 min intervals until change 
is <0.2 mv/min. Remove electrodes, blot lightly with ab- 
sorbent paper, and repeat reading with 0.2, 0.5, 2.0, and 10.0 
ppm std solns. Place electrodes in 0.2 ppm std soln until sam- 
ples are analyzed. (10 ppm std soln may be omitted if samples 
are known to contain <400 ppm F.) 

Plot potential (mv) on vertical arithmetic axis and F concn 
((jig/mL; ppm) on horizontal (logarithmic) axis of 2-cycle 
semilog graph paper. 

E. Preparation of Sample 

Dry foliage 48 hr at 80°. Grind to pass No. 40 sieve and 
store in clean, dry, tightly closed plastic bottle. Rotate bottle 
to mix sample thoroly before removing aliquots. 

F. Determination 

Accurately weigh ca 0.25 g powd sample, and place in 75- 
100 mL wide-mouth plastic container. Add 20 mL 0.057V HNO3 
and place on rotating shaker or stir mag. 20 min. Add 20 mL 
AN KOH (5.6 g/L) and agitate addnl 20 min. Add 5.0 mL 
Na citrate soln contg 1 ppm F, adjusted to pH 5.5, and 5.0 
mL 0.2jV HNO3. (Samples may be stored covered <4 hr at 
this point.) Det. mv readings as in 975. 04D and prep, cali- 
bration curve before and after each series of samples. 

If sample series contains mixt of high and low samples, 
make preliminary estn of F content after 2 min. Then det. F 
concn in samples contg <40 ppm first and in higher ones last. 

ppm F (p,g/g) = (C - 0.10) x 50/w 

where C = ppm F from curve; 0.10 = ppm background F in 
final soln; 50 = mL final soln; and w = g sample. 

Ref.: JAOAC58, 1129(1975). 

CAS-7782-41-4 (fluorine) 



984.02 Fluoride in Plants 
Willard-Winter Distillation Method 

Final Action 

See 944.08. 

978.03 Fluoride in Plants 
Semiautomated Method 

First Action 1978 
ASTM-lntersociety Committee-AOAC Method 

A. Principle 

Dried and ground plant material is ashed, fused with alkali, 
and dild to vol. In case of leaf samples, F on external surfaces 



Table 975.04 Preparation of Working Standard Solutions 

mL soln to be dild to 100 mL 



Concn, 


0.4 M 


0.8 M Na 100 ppm 10 ppm 


ppm 


KNO3 


citrate F soln F soln 


10 


10.0 


5.0 10.0 0.0 


2 


10.0 


5.0 2.0 0.0 


0.5 


10.0 


5.0 0.0 5.0 


0.2 


10.0 


5.0 0.0 2.0 


0.1 


10.0 


10.0 mL Na citrate soln contg 1 ppm F 



may be washed off sep. Digest and H 2 S0 4 are pumped into 
microdistn app. maintained at 170°. Stream of air carries acidi- 
fied sample to fractionation column where F and H 2 are distd 
into condenser, and condensate passes into small collector. 
Distillate is mixed continuously with alizarin F blue-lanthanum 
reagent, colored stream passes thru tubular rlowcell of color- 
imeter, and A is measured at 624 nm. 

Interfering metal cations and inorg. phosphate are not distd, 
and org. substances are destroyed by ashing. Interference from 
remaining volatile inorg. anions is reduced with high concn 
acetate buffer with some reduction in sensitivity. Very large 
arnts solid matter, particularly silicates, retard distn. There- 
fore, smallest sample consistent with obtaining suitable amt F 
should be used. Conditions must be carefully controlled, since 
accurate results depend upon obtaining same degree of effi- 
ciency of distn from samples as from std F solns used for cal- 
ibration. 

Acid concn during distn is maintained at const value by us- 
ing specific amts CaO and NaOH for ashing and fusion and 
HCIO4 for transfer of fused samples. Any marked change in 
vac. (>0.2" Hg or 5 torr) over short time indicates either leak 
or block in system. Distil at same vac. each day and maintain 
proper ratio between air flow on line drawing liq. and solid 
wastes from distn coil and on line drawing HF and H 2 vapor 
from distn unit (Fig. 978. 03 A). Adjust flowmeters to keep this 
ratio const and to maintain higher vac. on HF line, C i9 so that 
min. is diverted to waste line. 

Method can detect 0.1 |xg F/mL. Normal range is 0.1-4.0 
p,g F/mL. Dil. higher concns with NaOH-HC10 4 soln, (k). If 
digested samples routinely exceed 4.0 jmg/mL, modify anal. 
portion of pump manifold to reduce sensitivity, or use smaller 
sample aliquot (preferred). Most accurate results are obtained 
in middle or upper part of calibration curve. For example, to 
decrease sensitivity, pump sample thru 0.081" tube (2.5 mL/ 
min) and dil. with H 2 pumped thru 0.065" tube (1.6 mL/ 
min) before sample enters distn app. Total vol. sample and 
diluent should approx. original vol. used (4.1 mL/min). 

If air-borne contaminants are present in laboratory, attach 
small drying bulb contg CaC0 3 granules to air inlet tube of 
microdistn unit. Teflon distn coil of microdistn unit must be 
cleaned periodically to avoid accumulation of solids which re- 
duce sensitivity. 

Coefficient of variation of 20-100 ppm F is generally <10%. 
Samples with large amt of Si (orchard grass) or Al may present 
special difficulties. There should be no significant deviation 
from linearity with different amts sample and with different 
amts added F. 

B. Apparatus 

(Cat. Nos refer to current Technicon equipment, except where 

indicated. Corresponding equipment under previous Cat. Nos 

is satisfactory.) 

(a) Automatic analyzer. — (Fig. 978. 03B) Auto Analyzer, 
Technicon Instruments Corp., or equiv. (7) Sampler. — Sam- 
pler IV with rotary stirrer and 8.5 mL plastic sample cups. 
Use 10 or 20 /hr cam with 1:3 sample-to-wash ratio (No. 171- 



AOAC Official Methods of Analysis (1990) 



NONMETALS 53 




FIG. 978.03A— Schematic drawing of air flow system used in semiautomated analysis for fluoride 



AO 15-07). (2) Colorimeter. — With 15 mm tubular flowcell and 
624 nm interference filter (199-A001-05). (3) Recorder.— Ra- 
tio type with 2-100 mv full scale range (01 1-A1 15-01). (4) 
Multichannel proportioning pump and manifold cartridge. — 
With assorted pump tubes, nipple connectors, and glass con- 
nectors (pump III 113-A014-08; cartridge 116-8340-01). 

(b) Pulse suppressors. — For sample and color reagent 
streams. Coil 10' length of 0.035" id Teflon std tubing around 
2.5" diam. tube. Force outlet end into short length of 0.045" 
id Tygon tubing which is then sleeved with piece of 0.081" 
Tygon tubing. Slip sleeved end over "h" fitting which joins 
sample and reagent streams. (Pulse suppressor included with 
manifold cartridge.) 



(c) Voltage stabilizer.— 161- AQQ7-Q1 (also part of 199-A001- 
05). 

(d) Rotary vacuum and pressure pump. — With continuous 
oiler. 

(e) Micro distillation apparatus. — (Fig. 978. 03C) Major 
components are (Cat. Nos. are those of Lurex Scientific, ex- 
cept as noted); (Y) Bottom only of jacketed 1 L resin reaction 
flask with conical flange (JR-5130), modified by evacuating 
space between inner and outer walls and sealing off port (a); 
(2) resin reaction flask top with conical flange (JR-7935) mod- 
ified to have one ¥ 29/42 center joint and four ~§ 24/40 side 
joints; (3) resin reaction flask clamp (JR-92 10-0000); (4) vari- 
able high-speed stirrer (S-6362) (d); (5) stainless steel, heavy 



Tube Size 
(incnesl sampler 11 






(7) O 090 Wcter ( 2 90 ml/mi 




®o 



056 Water (I 20 ml/mm) 



-0= 



© 0090Somple (2 90 ml/mm) \ 



-Q> 



0035 Air (0 42 ml/mm) 



Microdistiilotion 
device 



© 



-®- 



© 0.081 At" 1 1250- 



Gioss Bead Mixer 



■— ®- 



"Qj) 008i Impinqer (250 ml/mm} 



-^ 



PULSE 

suppressors Waste -*- 



@ 0073(S) A-C 
_^\ Reagent ( I 69 ml/mm) 

— ®- 



0.045 (S) (0 70 ml /mm) 



Time Delay Coil 



Bubble 
trap 



Vacuum 
pump 



£ 



PROPORTIONING 
PUMP 



^y 



□ 



COLORIMETER RECORDER 

'5 mm Tubular f c 

624 m „ flUeti 



FIG. 978.03B — Flow diagram for semiautomated analysis for fluoride 



54 



Plants 



AOAC Official Methods of Analysis (1990) 




FIG. 978.03C— Schematic drawing of microdistillation appara- 
tus 

duty stirrer stuffing box with ¥ 29/42 and shredded Teflon 
packing (JS-1 160-0102 and JS-3050-0000); (6) 10 mm diam. 
stainless steel stirrer rod with propeller to fit stuffing box; (7) 
therm ometer-thermoregulator, range 0-200° (c); (8) electronic 
relay control box; (9) low drift immersion heater, 750 watts 
{b); (10) 30' length coil of flexible Teflon TFE tubing, Vs" id, 
3 /j 6 " od, 0.030" wall, on rigid support of such diam. that com- 
pleted coil will fit into resin reaction flask (avoid kinking of 
tubing) (<?); (77) 2 flowmeters with ranges 0. 15-1 .00 and 0.6- 
5.0 L/min, both with needle valve controls (Dwyer lnstru- 



Points pushed 
into glass 




This shoulder should be 
rounded, not abrupt 



6 mm od 



FIG. 978.03D— Microdistillation column 



ments, Inc., PO Box 373, Michigan City, IN 46360); (72) vac. 
gage with range 0-10" Hg or 0-254 torr (mm Hg); (73) frac- 
tionation column of borosilicate glass (g; see also Fig. 978. 03D; 
(14) distillate collector; (75) H 2 0-jacketed condenser (h); (16) 
Dow-Corning 200 fluid (100 centistokes at 25°) (/); and (17) 
condenser (j). 

(f) Crucibles.— Inconel, Ni, or Pt, 40-50 mL. 

(g) Airflow system. — (Fig. 978. 03 A) Draw air thru air inlet 
tube, (a), before Teflon microdistn coil, (b). Air sweeps thru 
(b) to fractionation column, and is diverted into 2 channels. 
In channel c f , air passes thru H 2 0-jacketed condenser, (d), 
sample trap, (<?), to waste bottle, (/). Air then passes thru Vs" 
id glass tube directed against surface of H 2 S0 4 in waste bottle, 
(g). Partially dehydrated air passes thru gas drying tower, (h), 
contg 450 g indicating silica gel. Emerging air passes thru T - 
tube, (/), connected to vac. gage, (j) (0-10" Hg or 0-254 
torr), thru flowmeter, (k) (0-5 L/min), thru T-tube, (/), and 
then to vac. pump, (m). 

In channel c 2 , air passes thru H 2 0-jacketed waste trap, (ri), 
to waste bottle, (o). Air leaving waste bottle flows thru drying 
bulb, (/?), filled with indicating silica gel, and the dry air then 
passes thru flowmeter, (q) (0-1 L/min). Air stream then con- 
nects thru T-tube, (/), with air from first channel. 

C. Reagents 

(Caution: See safety notes on perchloric acid and sulfuric 
acid.) 

(Deionized H 2 may be used. CaO for ashing and NaOH for 
fusion must be low in F.) 

(a) Sulfuric acid.— (1 + 1). Mix 500 mL H 2 S0 4 with 500 
mL H 2 and cool before use. 

(b) Acetate buffer.— 2 A4M (pH 4.0). Dissolve 60 g 
Na0Ac.3H 2 in 500 mL H 2 0, add 100 mL HOAc, and dil. 
to 1 L with H 2 0. Stable at 25°. 

(c) Alizarin fluorine blue color reagent stock soln. — 0.0 1M. 
Suspend 0,9634 g reagent (alizarin complexone, alizarin com- 
plexan; 3-amino-ethylalizarin-Af,/V-diacetic acid; Burdick & 
Jackson Laboratories, Inc.) in ca 100 mL H 2 in 250 mL vol. 
flask. Add 2 mL NH 4 OH and shake until completely dis- 
solved. Add 2 mL HOAc and dil. to vol. with H 2 0. Stable 
indefinitely at 4°. 

(d) Lanthanum nitrate stock soln. — 0.02A7. Dissolve 8.6608 
g La(N0 3 ) 3 .6H 2 in H 2 and dil. to 1 L in vol. flask. 

(e) Wetting soln. — 30% soln (w/v) polyoxyethylene lauryl 
ether in H 2 (Brij-35, Technicon No. T21-0110). Soln is sta- 
ble at 25°. 

(f ) Working reagent. — Mix, in order listed, 300 mL acetate 
buffer, 244 mL H 2 0, 300 mL acetone, 100 mL /m-butanol, 
36 mL alizarin fluorine blue stock soln, 20 mL La (N0 3 ) 3 stock 
soln, and 2 mL wetting soln. Unused reagent is stable 7 days 
at 4°. Before using reagent, place under vac. 10 min to remove 
air bubbles from soln. 

(g) Fluoride std solns. — (7) Stock soln. — 100 jjig F/mL. 
Dissolve 0.2207 g NaF in H 2 and dil. to 1 L. (2) Working 
solns. — Prep. 7 solns contg 0.2, 0.4, 0.8, 1.6, 2.4, 3.2, and 
4.0 |xg F/mL. Before dilg to vol., add 6 g NaOH and 20 mL 
70% HC10 4 for each 100 mL final working soln so that stds 
have same composition as sample solns. Dil. with H 2 only 
for analysis of H 2 samples or air samples absorbed in H 2 0. 
Store working solns at 4° in polyethylene bottles; stable in 
presence of NaOH. 

(h) EDTA solns. — 1%. Dissolve I g Na 4 EDTA in 99 mL 
H 2 0. Prep. 0.05% and 0.01% solns by mixing 5 mL 1% soln 
with 95 mL H 2 and 1 mL 1% soln with 99 mL H 2 0, resp. 

(i) Phenolphthalein soln. — Dissolve 1 g phthln in 50 mL 
absolute alcohol or isopropanol and add 50 mL H 2 0. 



AOAC Official Methods of Analysis (1990) 



NONMETALS 



55 



(j) Detergent. — Alconox (Alconox, Inc., 215 Park Ave S, 
New York, NY 10003); available from laboratory supply firms. 

(k) Sodium hydroxide-perchloric acid soln. — Dissolve 6 g 
NaOH in H 2 0, add 40 mL 70% HC10 4 (1 + 1), and dil. to 100 
mL with H 2 0. Use to dil. samples when F in unknown sample 
exceeds std curve. 

D. Preparation of Sample 

(a) Leaves. — If it is necessary to remove surface F, wash 
sample with aq. soln contg 0.05% detergent and 0.05% 
Na4EDTA in polyethylene container 30 sec with gentle agi- 
tation. Remove, drain 3-4 sec, and rinse 10 sec in each of 3 
beakers of H 2 0. Discard solns after use. 

(b) Fresh plant tissues. — Dry 24-48 hr in 80° forced-draft 
oven, and grind as in (c). 

(c) Dry plant tissues. — Grind in semimicro Wiley mill to 
pass No. 40 sieve, and store in plastic container. 

E. Ashing and Fusion 

Accurately weigh 0.1-2.0 g well mixed dried plant tissue 
into crucible. Add 100±10 mg low-F CaO, enough H 2 to 
make loose slurry, and 4 drops phthln soln. Mix thoroly with 
polyethylene policeman. Final mixt. should be purple and re- 
main purple during evapn to dryness. 

Place crucible on cold hot plate and under IR lamp. Evap. 
under lamp to dryness, turn on hot plate, and char 1 hr. Trans- 
fer crucible to furnace at 600° and ash 2 hr. (Caution: To avoid 
flaming, place crucibles at front of furnace with door open ca 
5 min to further char samples; then reposition in furnace.) 

Remove crucibles, add 3.0 ±0.1 g NaOH pellets, and re- 
place in furnace with door closed to melt NaOH. (Caution: 
Avoid creeping of molten NaOH.) Remove crucibles individ- 
ually and swirl to suspend particulate matter until melt is par- 
tially solidified. Let cool until addn of small amt H 2 does 
not cause spattering. Wash down inner walls with 10-15 mL 
H 2 0. Suspend melt with polyethylene policeman and transfer 
with H 2 to plastic tube graduated at 50 mL. Rinse crucible 
with 20~.0 mL 70% HC10 4 (1 + 1), add rinse to tube, and dil. 
to 50 mL with H 2 0. Solns can be stored at this point if tightly 
capped. 

Analyze blank contg all reagents with each set of ca 10 
samples. 

Clean crucibles as soon as possible after each use. Boil In- 
conel crucibles I hr in 10% NaOH soln. Rinse with hot tap 
H?0, detergent, and then distd H 2 0. Immerse crucibles which 
held samples contg >100 (xg F in AN HC1 45 min before boil- 
ing in NaOH soln. Perform blank analyses on these crucibles 
before addnl use to check for contamination. Scrub Ni and Pt 
crucibles with detergent and hot H 2 and rinse thoroly with 
H 2 0. Briefly rinse crucibles which held samples contg >100 
fjLg F in 4 7V HC1 before rinsing with H 2 0. 

F. Analytical System 

Place F std solns, ashed and fused samples, or impinged air 
samples in 8.5 mL plastic cups in sample module. Actuate 
sampler and pump from cup at net rate of 2.48 mL/min with 
air segmentation of 0.42 mL/min after sampler crook, and pump 
into microdistn device thru sample inlet (7, Fig. 978.03C), us- 
ing 0.051" id Teflon tubing. Pump H 2 S0 4 at 2.5 mL/min thru 
acid inlet (m, Fig. 978. 03C). Cool and discard acid and solids. 
Pump distillate from sample trap at 2.0 mL/min thru 0.051" 
Teflon tubing, add color reagent at 1.69 mL/min, and mix in 
4" length of Vs" id glass tubing packed with pieces of 20 mesh 
broken Pyrex glass. Pass colored stream thru time delay coil 
of 15' of 0.035" Teflon spaghetti tubing, thru debubbler fitting 
where small portion of stream and bubbles are removed to waste 
bottle at rate of 0.70 mL/min, and thru 15 mm tubular flowcell 
of colorimeter. A is measured at 624 nm and plotted on re- 



corder. Lag time from sampling to appearance of peak is ca 
5 min. Time between samples is 6 min with sampling rate of 
10/hr and 3 min at 20/hr. 

G. Start-Up 

Turn on H 2 to condenser and cooling jacket. Turn on col- 
orimeter. Engage manifold on proportioning pump and start 
pump. Turn on stirring motor of microdistn unit, vac. pump 
adjusted for full vac, and heater of microdistn unit. Connect 
lines to H 2 S0 4 , color reagent, and H 2 bottles. Sampling tube 
of sampler unit should be in H 2 reservoir. Equilibrate app. 
until silicone oil in microdistn unit reaches 1.70 ±2°. Check that 
all connections are secure. Adjust distn flowmeter (k, Fig. 
978.03C) to 2.5-3 L/min; adjust waste flowmeter (q) to 0.3 
L/min. Distillate should now fill sample trap. Readjust flow- 
meter (k) to give reading on vac. gage of 5-6" Hg (127-150 
torr). (Satisfactory setting for app. must be detd by trial and 
error. Once detd, use each day.) No air bubbles should be in 
anal, system beyond point where color reagent and distillate 
streams are joined. Turn on recorder, adjust baseline to desired 
level, and run several min to assure that all components are 
operating properly. Baseline should be reasonably smooth and 
straight. 

Transfer F std solns to 8.5 mL plastic cups and place in 
sampler. Sep. last std soln from sample solns with cup of H 2 0. 
Program sampler for 10 samples/hr (90 sec sampling period, 
270 sec washout period) or 20 samples/hr (45 sec sampling 
period, 135 sec washout period). 

Prep, std curve, 978. 03 J, before and after each day's set of 
samples. Net A of 0.7-0.9 should be obtained with std soln 
contg 4 (xg F/mL. A of each std soln should be reproducible 
within 10% from day to day and std curve should be linear 
from 0.2 to >3.2 u.g/mL. 

H. Shut-Down 

Turn off chart recorder. Disconnect H 2 S0 4 line and place in 
H 2 0. Disconnect color reagent line and place in 0.01% EDTA 
soln ca 1 min; then transfer line to H 2 and let H 2 pass thru 
system ca 5 min. Clean Teflon distn coil as in 978.031(a). 
Turn off heater and stirrer of microdistn unit. Turn off vac. 
pump. Release pump tube manifold. Turn off H 2 to con- 
denser and cooling trap. 

/. Maintenance 

(a) Cleaning of Teflon distillation coil. — (After use with 
samples contg particulate matter.) Briefly insert Tygon tube 
connected to air inlet line of microdistn unit into 0.01% EDTA 
soln. After all deposited material has been removed, wash with 
3—4 five mL portions distd H 2 0. 

(b) Pump tubes. — Replace after 200 working hr or earlier 
if hard and inflexible or flattened. Always leave in relaxed po- 
sition when not in use. Remove dirt and grease from pump 
plates and rollers after each day of use. 

(c) Indicating silica gel. — Regenerate when ca 2 /s has lost 
normal blue color. 

(d) Cleaning tubing. — Clean tubing contg reagent after each 
daily run with 0.01% EDTA soln followed by distd H 2 0. 

(e) Monthly checks. — Oil proportioning pumps monthly. 
Check gain on recorder monthly and adjust. 

J. Calibration and Standards 

Before and after each day's set of samples, prep, std curve 
by transferring aliquots of each working std soln to 8.5 mL 
sample cups and proceed with analysis. Draw straight line con- 
necting baseline before and after analysis. Record A of each 
peak and subtracts of baseline at peak. Plot netv4 against jxg 
F/mL. 



56 



Plants 



AOAC Official Methods of Analysis (1990) 



K. Calculations 

ppm F in sample = (F x V X D)/W 
where F = (xg F/mL sample from std curve; V = mL sample, 
usually 50; D = diln factor used only when F of sample ex- 
ceeds std curve = mL final vol. to which original aliquot was 
dild with NaOH-H00 4 soln, (k)/mL original aliquot taken; 
and W = g sample taken for analysis. 

L. Check Procedure 

(a) Contamination. — Perform reagent and equipment blank 
with crucibles and reagents but without sample to detect con- 
tamination from previous samples, contaminated furnace, and 
reagents. Blank values >5 jxg F are evidence of contamina- 
tion. Perform 2 blank detns with each set of 20-40 samples. 
Usual blanks are 1-3 jxg. 

(b) Recoveries .- — Occasionally add known amts F std soln 
from microburet to aliquots of low F tissue. Recovery of added 
F should be 100± 10%. Low values indicate loss of F, possibly 
during pretreatment; high values indicate contamination. 

(c) Linearity. — Occasionally analyze different amts (0.1- 
2.0 g) plant sample contg 50-65 ppm F. Linear relationship 
should exist between F found and amt tissue taken. Nonline- 
arity may indicate that some component of tissue is retarding 
distn or interfering with color development. 

(d) Calibration curves. — Prep, at least twice daily. 

M. Trouble Shooting 

(a) Irregular baseline. — May result from: (1) excessive pulse 
pressures — check for faulty pump tubes, absence of surge sup- 
pressors, or improperly made or placed suppressors; (2) air 
bubbles in flowed . I — check for absence of debubbler bypass, 
blockage in reagent pump tube, or periodic emptying of sam- 
ple trap (latter results if air flow to distn trap becomes too 
great); (3) excessive H 2 S0 4 carry-over — check for too high 
temp, in oil bath, improper H 2 S0 4 concn, or too high vac. on 
system; (4) air flow imbalances — check flowmeter settings, 
trapped air in tubing, or leak or block in system; (5) high F 
content in samples (baseline may not return to normal between 
samples) — dil. or check sampling speed and sample-to-wash 
ratio. 

(b) Irregular peaks. — Asymmetrical or double peaks or peaks 
with shoulders may result from: (1) baseline irregularities; (2) 
interfering substances from sample or impure reagents; (3) in- 
adequate buffer concn; or (4) excessive amts solids in distn 
coil. Presence or accumulation of solids may be due to insuf- 
ficient flow of H 2 S0 4 , too large sample, excess CaO or NaOH 
in sample, inadequate suspension of particles in samples, or 
lack of proper air segmentation in sample tubing. 

(c) Poor reproducibility. — Check for: improper sample 
pickup; faulty pump tubes; inadequate washing of distn coil 
between samples; large deviations in acid concn, temp., or air 
flow in distn coil; or changes in vac. on waste system. 

Refs.: JAOAC 55, 991(1972); 61, 150(1978). 

CAS-7782-41 -4 (fluorine) 



966.01 Phosphorus in Plants 

Gravimetric Quinolinium Molybdophosphate Method 
Final Action 1974 

A. Preparation of Solution 

Accurately weigh ca 2 g plant sample in porcelain dish, and 
add 7.5 mL Mg(N0 3 ) 2 soln (dissolve 950 g P-free 
Mg(N0 3 ) 2 .6H 2 in H 2 and dil. to 1 L). Dry in oven 2 hr at 
110-115° (or until dry). Ignite carefully over Fisher burner, 
or equiv., until bubbling and smoking cease. Complete ashing 



in furnace 4 hr at 550-600°. Dissolve ash in few mL HO 
(2+1) and evap. to dryness on steam bath. Take up residue in 
10-15 mL HC1 (1 +9) and filter thru coarse paper into 200 mL 
vol. flask. Wash paper thoroly with FLO and let filtrate cool 
to room temp. Dil. to vol. with H 2 0. 

B. Determination 

Pipet 40 mL aliquot into 300 or 500 mL erlenmeyer and 
proceed as in 962. 02C. 

Ref.: JAOAC 49, 284(1966). 



933.01 • Phosphorus in Plants 

Macro Method 

Final Action 
Surplus 1974 



See 3.065, 13th ed. 



931.01 



Phosphorus in Plants 

Micro Method 

Final Action 



A. Reagents 

(a) Phosphorus std soln. — 0.025 mg P/mL. Dissolve 0.4394 
g pure dry KH 2 P0 4 in H 2 and dil. to 1 L. Dil. 50 mL of this 
soln to 200 mL. 

(b) Ammonium molybdate soln. — Dissolve 25 g NH 4 mo- 
lybdate in 300 mL H 2 0. Dil. 75 mL H 2 S0 4 to 200 mL and 
add to NH 4 molybdate soln. 

(c) Hydroquinone soln. — Dissolve 0.5 g hydroquinone in 
100 mL H 2 0, and add 1 drop H 2 S0 4 to retard oxidn. 

(d) Sodium sulfite soln. — Dissolve 200 g Na 2 S0 3 in H 2 0, 
dil. to 1 L, and filter. Either keep this soln well stoppered or 
prep, fresh each time. 

B. Preparation of Solution 

To 1 or 2 g sample in small porcelain crucible add 1 mL 
Mg(N0 3 ) 2 soln (dissolve 950 g P-free Mg(N0 3 ) 2 .6H 2 in H 2 
and dil. to 1 L), and place on steam bath. After few min, 
cautiously add few drops HC1, taking care that gas evolution 
does not push portions of sample over edge of crucible. Make 
2 or 3 further addns of few drops HC1 while sample is on bath 
so that as it approaches dryness it tends to char. If contents 
become too viscous for further drying on bath, complete drying 
on hot plate. Cover crucible, transfer to cold furnace, and ig- 
nite 6 hr at 500°, or until even gray ash is obtained. (If nec- 
essary, cool crucible, dissolve ash in little H 2 or ale. -glyc- 
erol, evap. to dryness, and return uncovered to furnace 4-5 
hr longer.) Cool, take up with HC1 (1+4), and transfer to 100 
mL beaker. Add 5 mL HC1 and evap. to dryness on steam 
bath to dehydrate Si0 2 . Moisten residue with 2 mL HC1, add 
ca 50 mL H 2 0, and heat few min on bath. Transfer to 100 mL 
vol. flask, cool immediately, dil. to vol., mix, and filter, dis- 
carding first portion of filtrate. 

C. Determination 

To 5 mL aliquot filtrate in 10 mL vol. flask add 1 mL NH 4 
molybdate soln, rotate flask to mix, and let stand few sec. Add 
1 mL hydroquinone soln, again rotate flask, and add 1 mL 
Na 2 S0 3 soln. (Last 3 addns may be made with Mohr pipet.) 
Dil. to vol. with H 2 0, stopper flask with thumb or forefinger, 
and shake to mix thoroly. Let stand 30 min, and measure A 
with spectrophtr set at 650 nm. Report as % P. 

Refs.: JAOAC 14, 216(1931). J. Biol. Chem. 59, 255(1924). 

CAS-7723-14-0 (phosphorus) 



AOAC Official Methods of Analysis (1990) 



NONMETALS 



57 



936.04* 



Selenium in Plants 

Gravimetric Method 

Final Action 
Surplus 1974 



(Applicable to materials contg >2 ppm Se) 
See 3.073, 11th ed. 



969.06 Selenium in Plants 

Fluorometric Method 

First Action 1969 
Final Action 1974 

{Caution: See safety notes on photofluoro meter, wet oxida- 
tion, nitric acid, and perchloric acid.) 

A. Apparatus 

(a) Micro-Kjeldahl flasks. — 30 mL Pyrex, ca 170 mm total 
length with ¥ 12/18 outer joint at mouth. 

(b) Air condensers . — 1 x 1 40 mm Pyrex tubes with IT 2 / 1 8 
inner joint. 

(c) Micro-Kjeldahl digestion unit with glass fume duct. — 
Fit rack to hold flasks and attached air condensers in nearly 
upright position during early stages of digestion. Use in fume 
hood. 

(d) Fluorometer. — Capable of illuminating sample at 369 
nm and measuring fluoresced light at 525 nm. Spectrofluo- 
rometer set to above wavelengths is also satisfactory. 

B. Reagents 

(Use deionized H 2 distd in glass for prepg solns and dilns.) 

(a) Nitric acid. — Redistd in glass. 

(b) Hydroxylamine-ethylenediaminetetraacetic acid soln.-— 
Add ca 20 mL H 2 to 1.9 g EDTA (acid form). Slowly add 
ca 5N NH 4 OH with stirring until EDTA just dissolves. Some 
excess NH 4 OH is not harmful. Dissolve 6 g NH 2 OH.HCl in 
100 mL H 2 0. Combine solns and diJ. to 250 mL with H 2 0. 

(c) Cresol red indicator. — Dissolve 0.1 g cresol red in 10 
mL H 2 and 1 drop 50% NaOH soln. Dil. to 50 mL with H 2 0. 

(d) Selenium std soln. —0.3 fig Se/mL. Add 10 mL HNO3 
to 30.0 mg Se (purity >99%) and warm to dissolve. Dil. to 
100 mL with H 2 0, mix well, and transfer exactly 1 mL to 
micro-Kjeldahl flask. Add 2 mL 70% HCIO4 and 1 glass bead. 
Boil gently to HCIO4 fumes and cool. Add 1 mL H 2 and 1 
mL HC1 (1+4); heat 30 min in boiling H 2 bath. Transfer to 
1 L vol. flask and dil. to vol. with ca \N HC1. Store in all- 
glass container. Soln is stable several months at room temp. 

(e) Decalin. — Eastman Kodak No. 1905 decahydronaph- 
thalene, or equiv. 

(f) 2 ,3-Diaminonaphthalene (DAN) soln. — Prep, soln in 
semidarkened room or in room with only yellow light at time 
of detn. Protect from light and prep, fresh for each set of detns. 
Add 50 mL ca 0.1 N HC1 to 0.05 g DAN (No. 13,653-0, Aid- 
rich Chemical Co.). Place in 50° H 2 bath in dark 15 min. 
Cool to approx. room temp, and ext twice with 10 mL decalin, 
shaking vigorously each time and discarding decalin. Filter thru 
paper satd with H 2 0. For >8 detns, prep, larger amt. 

C. Preparation of Samples 

Grind air-dried samples to pass No. 18 or finer sieve. Cut 
fresh or wet samples finely with scissors or knife, or grind in 
food chopper to assure representative sample. 



Some plants (e.g.. Astragalus hisulcatus, A. racemosus, 
Stanley a bipinnata, and Oonopsis condensatd) contain Se in 
volatile form that is lost during drying. Analyze these plants 
without drying. With usual agricultural crops, this is not a 
problem if drying is performed at 60-70°. 

D. Preparation of Fluorometric Blanks and Standard 

(a) Blank. — Place 1 mL H 2 in micro-Kjeldahl flask. (For 
samples contg <0.1 ppm, carry 10 mL HN0 3 as blank thru 
entire detn.) 

(b) Std. — Place 1.0 mL Se std soln in micro-Kjeldahl flask. 

Add 2 mL 70% HC10 4 to each flask and continue as in detn, 
beginning "Mix contents of flasks ..." 

E. Determination for Low Level Samples 

For samples containing <4 ppm selenium. — Weigh <l g 
sample (air-dried wt basis) contg <0.4 |mg Se into micro-Kjel- 
dahl flask. Add 1 glass bead, previously cleaned with HNO3. 
Add 10 mL HN0 3 and let stand at room temp. >4 hr. (Use 5 
mL HNO3 for samples <0.5 g.) Affix air condenser and place 
flask in nearly upright position on micro-Kjeldahl digestion 
unit. Heat ca 15 min with low flame and then increase heat 
until HNO3 condenses in lower part of condenser. Heat 10 min 
longer, turn off burner, and let cool 5 min. Wash down sides 
of flask with 2 mL 70% HCIO4 thru air condenser. Swirl flask 
and continue refluxing 15 min. Remove condenser and con- 
tinue heating, drawing off fumes in fume duct, until HCIO4 
fumes appear and then 15 min longer. Cool, add 1 mL H 2 0, 
and again heat to HCIO4 fumes and 1-2 min longer. Cool, 
and add 1 mL H 2 0. 

Mix contents of flasks and add 1 mL HC1 (1+4) to each. 
Place in boiling H 2 bath 30 min. Cool to ca room temp. 

To each flask add 5 mL NH 2 OH-EDTA soln and 2 drops 
cresol red indicator. Neutze to yellow with ca 5 N NH 4 0H and 
add HC1 (1+4) to orange-pink. From this point, perform all 
operations in semidarkened room or room with yellow light 
only. Prep. DAN soln, add 5 mL to each flask, and dil. to 
neck with ca 0.1 TV HC1. Mix and place in 50° H 2 bath in 
dark 25 min. 

Remove flasks from H 2 bath and cool to ca room temp, 
in pan of H 2 0. Pour solns into 125 mL separators with Teflon 
stopcocks and contg 10.0 mL decalin. Shake vigorously >30 
sec, let stand ca 1 min, and drain and discard lower layer. 
Wash decalin twice by shaking vigorously >15 sec with 25 
mL ca 0.1 TV HC1. (VirTis, Rt 208, Gardiner, NY 12525, Ex- 
tractomatic shaker with 100 mL separators may be substituted. 
When used, shake ext 5 min and wash I min periods.) Trans- 
fer decalin layer to 12 mL centrf. tubes and centrf. 2 min at 
moderate speed. Pour decalin soln into fluorometer tubes, zero 
fluorometer against decalin, and read all tubes at 525 nm within 
5 min. Correct std and unknown readings for blank. 

ppm Se — 0.3 x sample reading/std reading x g sample 

F. Determination for Higher Level Samples 

For samples containing >4ppm selenium . — Proceed as above , 
thru second par. Dil. digest to adequate vol. and take aliquot 
contg ca 0.3 jxg Se for detn. Alternatively, digest sample in 
10 vols HNO3 2 hr on steam bath. Dil. to definite vol., and 
carry appropriate aliquot thru detn. Latter method is especially 
applicable when proper sampling requires large sample. Do 
not dil. decalin soln contg piazselenol, as this introduces er- 
rors. 

Ref.: JAOAC 52, 627(1969). 

CAS-7782-49-2 (selenium) 



58 



Plants 



AOAC Official Methods of Analysis (1990) 



920.10 



Sulfur in Plants 

Sodium Peroxide Method 

Final Action 



(Caution: See safety notes on sodium peroxide.) 

A. Preparation of Solution 

Place 1.5-2.5 g sample in ca 100 mL Ni crucible and add 
5 g anhyd. Na 2 C0 3 . Mix thoroly, using Ni or Ptrod, and mois- 
ten with ca 2 mL H 2 0. Add Na 2 2 , ca 0.5 g at time, mixing 
thoroly after each addn, and continue until mixt. becomes nearly 
dry and quite granular (ca 5 g Na 2 2 ). Place crucible over S- 
free flame or elec. hot plate and heat carefully, stirring oc- 
casionally, until contents are fused. (If material ignites, detn 
is worthless.) 

After fusion, remove crucible, let cool somewhat, and cover 
hardened mass with more Na 2 2 to depth of ca 5 mm. Heat 
gradually and finally with full flame until fusion again takes 
place, rotating crucible occasionally to bring any particles ad- 
hering to sides into contact with oxidizing material. Continue 
heating 10 min after fusion is complete. Cool somewhat, place 
warm crucible and contents in 600 mL beaker, and carefully 
add ca 100 mL H 2 0. After initial violent action ceases, wash 
material out of crucible, make slightly acid with HO (adding 
small portions at time), transfer to 500 mL vol. flask, cool, 
dil. to vol., and filter. 

B. Determination 

Dil. aliquot of prepd soln to ca 200 mL with H 2 and add 
HC1 until ca 0.5 mL free acid is present. Heat to bp and add 
10 mL 10% BaCl 2 soln dropwise with constant stirring. Con- 
tinue boiling ca 5 min, and let stand >5 hr in warm place. 
Decant thru ashless paper or ignited and weighed gooch. Add 
15-20 mL boiling H 2 to ppt, transfer to filter, and wash with 
boiling H 2 until filtrate is Cl-free. Dry ppt and filter, ignite, 
and weigh as BaS0 4 . Wt ppt x 0.1374 - S. 

Refs.: USDA Bur. Chem. Bull. 105, p. 151; 116, p. 92; 137, 
p. 30. 

CAS-7704-34-9 (sulfur) 



923.01 Sulfur in Plants 

Magnesium Nitrate Method 
Final Action 

A. Preparation of Solution 

Weigh 1 g sample into large porcelain crucible. Add 7.5 
mL Mg(N0 3 ) 2 soln (dissolve 950 g P-free Mg(N0 3 ) 2 .6H 2 in 
H 2 and dil. to 1 L), so that all material comes in contact with 
soln. (It is important that enough Mg(N0 3 ) 2 soln be added to 
ensure complete oxidn and fixation of the S present. For larger 
samples and for samples with high S content, proportionally 
larger vol. of this soln must be used.) Heat on elec. hot plate 
(180°) until no further action occurs. Transfer crucible while 
hot to furnace (<500°) and let it remain until sample is thoroly 
oxidized. (No black particles should remain. If necessary, break 
up sample and return to furnace.) Remove crucible and let cool. 
Add H 2 0; then HO in excess. Bring soln to boil, filter, and 
wash thoroly. If preferred, transfer soln to 250 mL vol. flask 
before filtering and dil. to vol. with H 2 0. 

B. Determination 

Dil. entire filtered soln, 923.01A, to 200 mL, or take 100 
mL aliquot of the measured vol., dil. to 200 mL, and proceed 
as in 920.10B. 



Ref.: JAOAC 6, 415(1923). 



OTHER CONSTITUENTS 

931.02 Sugars in Plants 

Preparation of Sample 
Final Action 

A. Preparation of Solution 

(a) General method. — Prep, fresh sample as in 922.02(b). 
Pour ale. soln thru filter paper or extn thimble, catching filtrate 
in vol. flask. Transfer insol. material to beaker, cover with 
80% alcohol, warm on steam bath 1 hr, let cool, and again 
pour ale. soln thru same filter. If second filtrate is highly col- 
ored, repeat extn. Transfer residue to filter, let drain, and dry. 
Grind residue so that all particles will pass thru 1 mm sieve, 
transfer to extn thimble, and ext 12 hr in Soxhlet app. with 
80% alcohol. Dry residue and save for starch detn. Combine 
ale. filtrates and dil. to vol. at definite temp, with 80% al- 
cohol. 

For dried materials, grind samples finely, and mix well. 
Weigh sample into beaker, and continue as above, beginning 
"... cover with 80% alcohol, ..." 

(b) Applicable when starch is not to be determined, — Prep. 
fresh sample as in 922.02(b), but boil on steam bath 1 hr. 
Decant soln into vol. flask, comminute solids in high-speed 
blender with 80% alcohol. Boil blended material on steam bath 
0.5 hr, cool, transfer to vol. flask, dil. to mark with 80% al- 
cohol at room temp., filter, and take aliquot for analysis. 

Grind dry material to pass No. 20 sieve or finer, transfer 
weighed sample to vol. II ask, and add 80% alcohol and enough 
CaC0 3 to neutze any acidity. Boil 1 hr on steam bath, cool, 
adjust vol. at room temp, with 80% alcohol, filter, and take 
aliquot for analysis. 

B. Clarification with Lead (1) 

Place aliquot ale. ext in beaker on steam bath and evap. off 
alcohol. Avoid evapn to dryness by adding H 2 if necessary. 
When odor of alcohol disappears, add ca 100 mL H 2 and 
heat to 80° to soften gummy ppts and break up insol. masses. 
Cool to room temp, and proceed as in (a) or (b): 

(a) Transfer soln to vol. flask, rinse beaker thoroly with H 2 0, 
and add rinsings to flask. Add enough said neut. Pb{OAc) 2 
soln to produce flocculent ppt, shake thoroly, and let stand 15 
min. Test supernate with few drops of the Pb(OAc) 2 soln. If 
more ppt forms, shake and Jet stand again; if no further ppt 
forms, dil. to vol. with H 2 0, mix thoroly, and filter thru dry 
paper. Add enough solid Na oxalate to filtrate to ppt all the 
Pb, and re filter thru dry paper. Test filtrate for presence of Pb 
with little solid Na oxalate. 

(b) Add twice min. amt of satd neut. Pb(OAc) 2 soln re- 
quired to cause complete pptn, as found by testing portion of 
supernate with few drops dil. Na oxalate soln. Let mixt. stand 
only few min; then filter into beaker contg estd excess of Na 
oxalate crystals. Let Pb ppt drain on filter and wash with cold 
H 2 until filtrate no longer gives ppt in oxalate soln. Assure 
excess of oxalate by testing with 1 drop Pb(OAc) 2 . Filter and 
wash pptd Pb oxalate, catching filtrate and washings in vol. 
flask. Dil. to vol. with H 2 and mix. 

C. Clarification with ton-Exchange Resins (2) 

Place aliquot ale. ext, 931. 02A, in beaker and heat on steam 
bath to evap. alcohol. Avoid evapn to dryness by adding H 2 0. 
When odor of alcohol disappears, add ca 15—25 mL H 2 and 
heat to 80° to soften gummy ppts and break up insol. masses. 
Cool to room temp. Prep, thin mat of Ceiite on filter paper in 
buchner or on fritted glass filter and wash until H 2 comes 
thru clear. Filter sample thru Ceiite mat, wash mat with H 2 0, 
dil. filtrate and washings to appropriate vol. in vol. flask, and 
mix well. 



AOAC Official Methods of Analysis (1990) 



Other Constituents 



59 



Place 50.0 mL aliquot in 250 mL erlenmeyer; add 2 g Am- 
berlite I R- 120(H) analytical grade cation (replaced by REXYN 
101(H) resin, Fisher Scientific Co.) and 3 g Duolite A-4(OH) 
anion ion exchange resins. Let stand 2 hr with occasional 
swirling. Take 5 mL aliquot de ionized soln and det. reducing 
sugars as glucose as in 959. 11B. 

Refs.: (1) JAOAC 14, 73, 225(1931); 15, 71(1932). 
(2) JAOAC 36, 402(1953). 



933.02 



See 959.11B. 



Glucose in Plants 

Micro Method 
Final Action 1965 



960.06 Fructose in Plants 

Somogyi Micro or Munson-Walker Method 

First Action 1960 
Final Action 1961 

A. Reagents 

(a) Glucose oxidase preparation. — Add slowly, stirring 
constantly, 100 mL H 2 to 5 g glucose oxidase prepn ("DeeO 
L-750" code 4633000, Miles Laboratories, Inc., 1127 Myrtle 
St, PO Box 70, Elkhart, IN 46514). Stir ca 1 min and centrf. 
or filter to obtain clear soln. Add ca 1 mL CHC1 3 and refrig- 
erate. Soln is stable >:! month. 

(b) Mcllvaine' $ citrate-phosphate buffer. — Dissolve 214.902 
g Na 2 HP0 4 .12H 2 and 42.020 g citric acid in H 2 and dil. to 
1 L. 

B. Determination 

To suitable aliquot add V-i its vol. of buffer to give pH ca 
5.8. Add 30% as much glucose oxidase prepn as estd glucose 
content (for 500 mg glucose add 150 mg glucose oxidase, i.e., 
3 mL soln), and few drops 30% H2O2 (omit if Somogyi method 
is to be used in detn). Let stand overnight at room temp. 

Det. fructose by Somogyi micro method, 959.1 IB, or by 
Munson-Walker method, 906.03, using Table 960.06. Check 
equivs in range of interest, using pure fructose as std, and cor- 
rect as necessary. 

Refs.: JAOAC 41, 307, 681(1958); 42, 650(1959); 43, 
512(1960); 44, 267(1961). 



921.03* Sugars (Reducing) in Plants 

Quisumbing-Thomas Method 

Final Action 
Surplus 1970 

See 31.048-31.049, 11th ed. 



930.07 



Sucrose in Plants 

Inversion Methods 

Final Action 



A. Hydrochloric Acid inversion 

Using aliquot of cleared soln, 931.02B, proceed as in 925.05. 

B. invertase inversion 

(a) For plants giving hydrolysis end point within 2 hours. — 
Pipet aliquot of cleared soln, 931. 02B, into 400 mL Pyrex 
beaker and make slightly acid to Me red with HO Ac. Add 3 
drops 1 % soln of Wallerstein invertase scales. Let mixt. stand 
at room temp. 2 hr. Add reagents as in 923. 09B, and det. 
reducing power. Calc. results as invert sugar. Deduct reducing 
power of original soln, also expressed as invert sugar, and 
multiply difference by 0.95. 

(b) For plants giving slower hydrolysis end point. — Place 
aliquot of soln, 931.02B, in small vol. flask. Make slightly 
acid to Me red with HO Ac. Add 3 drops \% soln of Wall- 
erstein invertase scales and few drops toluene. Stopper flask 
and let stand overnight or longer at room temp. Dil. to vol. 
with H 2 and use aliquot for reducing power as above. 

CAS-57-50-1 (sucrose) 



930.09 



See 920.39B. 



930.10 



See 962.09 



Ether Extract of Plants 

Gravimetric Method 

Final Action 



Fiber (Crude) in Plants 

Digestion Method 

Final Action 



906.01 Sugars (Reducing) in Plants 

Munson-Walker General Method 
Final Action 

See 906.03. 

Table 960.06 Abbreviated Munson and Walker Table for Cal- 
culating Fructose 

(From Official and Tentative Methods of Analysis, AOAC, 5th Ed., 1940) 



Cuprous Oxide, 


Fructose, 


Cuprous Oxide, 


Fructose, 


mg 


mg 


mg 


mg 


10 


4.5 


300 


148.6 


50 


23.5 


350 


174.9 


100 


47.7 


400 


201.8 


150 


72.2 


450 


229.2 


200 


97.2 


490 


253.9 


250 


122.7 


— 


~ 



978.04 Nitrogen (Total) (Crude Protein) 

in Plants 
Kjeldahl Methods 

First Action 1976 
Final Action 1978 

A. Kjeldahl Method for Nitrate-free Samples 
See 955.04. 

B. Kjeldahl Method for Nitrate-Containing Samples 
See 968.01. 



977.02 Nitrogen (Total) (Crude Protein) 

in Plants 



First Action 1977 



A. Automated Method 
See 976.05. 



60 



Plants 



AOAC Official Methods of Analysis (1990) 



B. Semiautomated Method 
See 976.06. 



948.02 



Starch in Plants 
Titrimetric Method 
Final Action 1965 



A. Reagents 

(a) Iodine-potassium iodide soln. — Grind 7.5 g 1 and 7.5 g 
KI with 150 mL H 2 0, dil. to 250 mL, and filter. 

(b) Alcoholic sodium chloride soln.— Mix 350 mL alcohol, 
80 mL H 2 0, and 50 mL 20% NaCl soln, and dil. to 500 mL 
with H 2 0. 

(c) Alcoholic sodium hydroxide soln. — 0.257V. Mix 350 mL 
alcohol, 100 mL H 2 0, and 25 mL 5/V NaOH, and dil. to 500 
mL with H 2 0. 

(d) Dilute hydrochloric acid. — 0.77V. Dil. 60 mL HC1 to 1 
L with H 2 0. 

(e) Somogyi phosphate sugar reagent. — Dissolve 56 g an- 
hyd. Na 2 HP0 4 and 80 g Rochelle salt in ca 1 L H 2 0, and add 
200 mL 1.00/V NaOH. Then slowly add, with stirring, 160 
mL 10% CuS0 4 .5H 2 soln. Dissolve 360 g anhyd. Na 2 S0 4 in 
this soln, transfer to 2 L vol. flask, and add exactly 200 mL 
QAN KI0 3 soln (3.5667 g/L). Dil. to vol., mix well, let stand 
several days, and filter thru dry paper into dry flask, discarding 
first 50 mL filtrate. Store reagent at 20-25°. It is 0.01N with 
respect to KI0 3 ; 5.00 mL is equiv. to 10 mL 0.005W Na 2 S 2 3 . 

Det. glucose factor of reagent as follows: Accurately weigh 
150 mg NBS glucose SRM into 1 L vol. flask, dissolve in 
H 2 0, dil. to vol., and mix well. Transfer 5 mL aliquot to 25 
x 200 mm Pyrex test tube, add exactly 5 mL Somogyi re- 
agent, stopper with size 00 crucible, and heat (together with 
several blanks contg 5 mL H 2 and 5 mL reagent) exactly 15 
min in boiling H 2 bath. Titr. as in detn. From difference 
between blank and std titrns, calc. mg glucose equiv. to 1 mL 
exactly 0.005/V Na 2 S 2 3 . Effective range for detn is 0.05-1.0 
mg glucose in 5 mL aliquot. 

(f) Sodium thiosulfate std soln. — 0.005 jV. Dissolve 2.73 g 
Na 2 S 2 3 .5H 2 in H 2 and dil. to 2 L. Stdze daily as follows: 
Add 1 mL KI soln, (g), and 3 mL 1.5 N H 2 S0 4 to 5 mL Som- 
ogyi sugar reagent. Let stand 5 min, and titr. with Na 2 S 2 3 
soln, adding starch indicator, (h), just before end point. 

(g) Potassium iodide soln. — 2.5%. Stabilize with little 
Na 2 C0 3 . 

(h) Starch indicator. — Make 1.5 g sol. starch into paste with 
few mL H 2 0, and add slowly, with stirring, to 300 mL boiling 
H 2 0. 

B. Determination 

Select sample as in 922.01, remove all foreign matter, dry, 
and grind to pass No. 80 sieve. Accurately weigh 0.1-1.0 g 
powd sample contg ca 20 mg starch into 25 x 1 50 mm Pyrex] 
test tube. Add ca 200 mg fine sand and 5 mL H 2 0, and mix 
well with stirring rod to wet sample. Heat tube in boiling H 2 
bath 15 min to gelatinize starch. Cool to room temp., and place 
in 22-25° bath. Add 5 mL 60% HC10 4 rapidly with const ag- 
itation. Grind tissue against lower wall of tube with stirring 
rod for approx. min at time. Repeat grinding frequently during 
30 min; then without delay, transfer quant, to 100 mL vol. 
flask with H 2 0. Add 3 mL 5% uranyl acetate soln to ppt pro- 
tein, dil. to vol. with H 2 0, mix well, and centrf. portion of 
mixt. Pipet 10 mL clear supernate into 25 X 150 mm test 
tube. Add ca 100 mg Celite, 5 mL 20% NaCl soln, and 2 mL 
I-KI reagent, and mix well. Let stand overnight, centrf., and 
decant. 

Wash starch-I ppt by suspending it in 5 mL ale. NaCl soln, 



centrf., and decant. Add 2 mL ale. NaOH soln to packed ppt. 
Gently shake and tap tube until ppt is no longer blue. (Do not 
use stirring rod; allow ample time for complex to decompose.) 
Wash walls of tube with 5 mL ale. NaCl soln, centrf. liberated 
starch, and wash with 5 mL ale. NaCl soln as before. Add 2 
mL 0.7 N HC1 to ppt. Stopper tube loosely with size 00 cru- 
cible, and heat 2.5 hr in boiling H 2 bath. (Bath should have 
cover with holes to accommodate tubes; holes not occupied by 
tubes must be covered.) Cool, and transfer quant, to 25 mL 
vol. flask. Add drop phenol red, 941. 17A, and neutze with 
1 N NaOH. Discharge color with 0.1 N oxalic acid, dil. to vol., 
and mix well. Transfer 5 mL aliquot to 25 X 200 mL Pyrex 
test tube, add exactly 5 mL Somogyi reagent, and stopper tube 
with size 00 crucible. Heat together with several blanks contg 
5 mL H 2 and 5 mL Somogyi reagent in vigorously boiling 
H 2 bath exactly 15 min. Remove tube from bath and cool to 
25-30°. Add 1 mL 2.5% KI soln down wall of tube without 
agitation and then add 3 mL 1.5 7V H 2 S0 4 rapidly with agita- 
tion. After all Cu 2 dissolves, titr. soln with 0.005 N Na 2 S 2 3 , 
adding starch indicator, (h), just before end point is reached. 
Treat blank solns similarly. 

% Starch = |'50(mL blank - mL sample) x 0.90/mg sample] 

x (jV/0.005) X G x 100 

where 50 = diln factor, 0.90 = factor glucose to starch, N — 
actual normality Na 2 S 2 3 soln, and G = mg glucose equiv. to 
1 mL 0.005 Af Na 2 S 2 6 3 . 

Refs.: Anal. Chem. 20, 850(1948). JAOAC 39, 423(1956). 



932.01 



Lignin in Plants 

Direct Method 
Final Action 1965 



A. Preparation of Sample 

Grind sample in mill to pass No. 80 sieve and dry at 105°. 
Ext weighed sample (5-10 g) 30 hr in Soxhlet app. with al- 
cohol-benzene soln (32 parts alcohol and 68 parts benzene by 
wt). Dry material in oven to free it from solvs and place in 
flask of suitable size. Add 150 mL H 2 0/g sample, and reflux 
3 hr. Filter mixt, while still hot, preferably thru weighed fritted 
glass crucible, and transfer extd material to flask. Add 1% HO 
(111 g coned HC1 + 3890 mL H 2 0) in proportion of 150 mL 
acid soln/g plant material, and reflux 3 hr. Filter mixt. while 
still hot thru fritted glass crucible previously used, wash with 
H 2 until acid-free, dry at 105°, and weigh. Calc. % total loss 
due to successive extn with alcohol-benzene soln, hot H 2 0, 
and 1% HC1. (With samples not especially rich in carbohy- 
drates and proteins, extn with hot H 2 may be omitted.) 

B. Apparatus 

App., Fig. 932.01, consists of: (1) 1 .5 L bottle, A, to which 
is attached by 2-hole rubber stopper 250 mL dropping funnel, 

C. having lower end of stem bent as illustrated and placed 
close to bottom of A; (2) Drechsel gas-washing bottle, D; (3) 
3 Pyrex test tubes, 38 X 300 mm diam., G, G f , G", connected 
in parallel by device, O, and immersed in wooden box, L, 
filled with crushed ice, //; and (4) bottle contg H 2 for ab- 
sorption of excess HC1, K. G, G\ and G" are provided with 
2-hole rubber stoppers; glass tube with right angle bend ex- 
tends thru 1 hole nearly to bottom of test tube, and similar 
tube extending ca 10 mm into test tube passes thru other hole. 
Rubber connections and stopcocks for regulating flow of gas 
are provided as indicated in diagram. A is filled with ca 500 
mL H 2 S0 4 and C with HO; HO flowing thru stopcock B into 
A generates HO gas, which is dried by H 2 S0 4 in D, and flows 
into G, G' , and G" contg samples and fuming HO reagent. 



AOAC Official Methods of Analysis (1990) 



Other Constituents 



61 





FIG. 932.01 — Apparatus for determining Hgnin 



C. Reagent 

Fuming hydrochloric acid. — (Caution: See safety notes on 
fuming acids.) Density 1 .212-1.223 at 15°. To 500 g NaCl in 
1 L g-s Pyrex distg flask, add cold soln of 250 mL H 2 in 
450 mL H 2 S0 4 . Connect side tube of distg flask to glass tube 
passing thru H 2 S0 4 wash bottle, and connect outlet tube of 
H 2 S0 4 wash bottle to another glass tube, immersed in flask 
contg 3 L HO. Surround flask contg HC1 with crushed ice. 
Heat distg flask with small flame and pass HC1 gas into acid 
soln until it attains sp gr of 1.212-1.223 at 15°. Keep reagent 
refrigerated at <0°. If only few detns are to be made, prep. 
correspondingly smaller amt. 

D. Determination 

Weigh three 1 g samples of extd and dried sample in weigh- 
ing bottle and place in 3 large test tubes, G, G\ and G". Add 
20 mL of the reagent to each tube, using this acid to wash 
down any particles clinging to sides. When all material is wet 
with reagent, add addnl 30 mL reagent. Add ca 3 drops capryl 
alcohol to minimize foaming. Place test tubes, G, G', and G", 
in wooden box, L, and surround with crushed ice. Lubricate 
tubes F, F\ and F" with drop of glycerol so that they move 
easily thru holes in rubber stoppers. Lead dry HC1 gas from 
generator into reaction mixts thru tubes F, F' , and F" (F' and 
F" are shown in top view), which reach nearly to bottom of 
tubes G, G\ and G" . Regulate flow of gas thru reaction mixts 
in G, G\ and G" by stopcocks shown in top view, continuing 
passage of gas 2 hr. (At first rather slow stream of gas passes 
in, but during last 15 min, flow is fairly rapid.) 

After reaction period, discontinue flow of gas, and discon- 
nect long tubes F, F\ and F" and outlet tubes of test tubes G, 
G\ and G" from O and P. Pull tubes F, F r , and F" just above 
surface of reaction mixt., and close with short pieces of rubber 
tubing having one end plugged with short piece of glass rod. 
Similarly close off outlet tubes, N, N f , and N fr . Place tubes 
contg reaction mixt. in cold room or refrigerator (8-10°) 24 
hr. 

Transfer contents of G ( G\ and G" to 1 L erlenmeyers, tak- 
ing care to remove any material adhering either to inside or 



outside of tubes F, F' , and F". Dil. reaction mixts to 500 mL 
with H 2 0. Connect flasks to reflux condensers and boil 1 hr. 
Prep. 3 gooches in usual manner, dry at 105°, and weigh. Ig- 
nite one of weighed crucibles, X, over Bunsen burner, cool in 
desiccator, and reweigh. Let contents of flasks cool to room 
temp, and filter thru weighed gooches. Wash ppts collected in 
gooches with hot H 2 0, dry at 105°, and weigh in weighing 
bottles. Ignite crude lignin in crucible X over Bunsen flame 
and det. wt ash. Place one of other 2 gooches in wide-neck 
Kjeldahl flask and det. % N in crude lignin as in 955.04. If 
methoxyl in lignin is to be detd, collect ppt from one of flasks 
in dried (105°) fritted glass crucible and proceed as in 956.07C. 

Wt lignin 
= wt crude lignin - wt ash — wt crude protein (N x 6.25) 

Calc. % lignin in original dry unextd material. 

Refs.: JAOAC 15, 124(1932); 18, 386(1935); 19, 107(1936). 

CAS-9005-53-2 (lignin) 



949.04 



Lignin in Plants 

Indirect Method 

Final Action 1965 



(Caution: See safety notes on asbestos.) 

Ext 1 g sample with alcohol-benzene (1+2) 4 hr in Soxhlet 
or comparable app. (extn vessel may be either coarse porosity 
Alundum or paper thimble, closed at top with filter paper or 
plug of cotton). Wash sample in thimble with suction, using 
2 small portions alcohol followed by 2 small portions ether. 
Heat at 45° in nonsparking oven to drive off ether, and transfer 
sample to 250 mL wide-mouth erlenmeyer. Add 40 mL 1% 
soln of pepsin in 0.1 N HCl, wetting sample well by adding 
small portion reagent, stirring or shaking thoroly, and finally 
washing down sides of flask with remaining soln. Incubate at 
40° overnight. 

Add 20-30 mL hot H 2 and filter, using filter stick. (Filter 



62 



Plants 



AOAC Official Methods of Analysis (1990) 



sticks are made with Pyrex fritted glass disk, 30 mm diaim, 
medium porosity. Thin layer of pre-ashed diat. earth (Hyflo 
Super-CeJ, or similar filter-aid) is sucked onto disk from H 2 
suspension. This is usually enough for easy filtration; if not, 
add extra Super-CeJ to material being filtered. Some sticks fil- 
ter slowly with some samples. It is advisable to obtain more 
than needed and discard slow-filtering ones. It is convenient 
to arrange filter sticks in set of 12 attached to vac, manifold 
by rubber tubing.) 

Repeat washing twice and then wash residue into flask by 
forcing 7-8 mL 5% (w/w) H 2 S0 4 downward thru filter stick, 
using air pressure. Wash stick further with the H 2 S0 4 , finally 
adding enough to bring total vol. to ca 150 mL. Reflux vig- 
orously on hot plate 1 hr, adding H 2 occasionally to maintain 
original vol. Filter off acid. Wash residue with three 20-30 
mL portions hot H 2 0, two 15—20 mL portions alcohol, and 
two 1.5 mL portions ether. Leave vac. on tew min to dry res- 
idue, and transfer from stick to flask by tapping and brushing. 
Heat to drive off any residual ether. If disk formed upon drying 
is difficult to break up into finely divided state (sometimes in 
case of immature plant samples), disperse residue in ether in 
flask and then boil off ether on steam bath. Add 20 mL 72% 
(w/w) H 2 S0 4 at 20° to residue and hold 2 hr at 20°, stirring 
occasionally. Add 125 mL H 2 0, filter, wash once with 20 mL 
hot H 2 0, and filter again. Wash residue from filter stick and 
reflux as before 2 hr, using 150 mL 3% (w/w) H 2 S0 4 . Filter 
residue onto gooch with asbestos pad and wash with hot H 2 
until acid-free. Dry at 105-110° and det. lignin by loss in wt 
on ignition at 600°. 

Ref.: JAOAC 32, 288(1949). 

CAS-9005-53-2 (lignin) 



Weigh 1-5 g into mortar and add ca 0. 1 g CaC0 3 or Na 2 C0 3 . 
Macerate tissue with pestle, add quartz sand, and grind short 
time; then add 85% acetone, little at time, and continue grind- 
ing until tissue is finely ground. Transfer mixt. to funnel, filter 
with suction, and wash residue with 85% acetone. Return res- 
idue to mortar with more 85% acetone and grind again. Filter 
and wash as before. Repeat procedure until tissue is devoid of 
any green, and washings are colorless. (It is advisable to grind 
residue at least once with undild acetone and then to add enough 
H 2 at end to bring acetone concn to 85%. High-speed blender 
may be used instead of mortar to macerate and ext tissue (see 
942. 04C), but each investigator should satisfy himself that de- 
vice used exts tissue completely.) When extn is complete, 
transfer filtered ext to vol. flask of appropriate size and dil. 
to vol. 

Measure T of soln with photoelec. colorimeter, and read amt 
of chlorophyll present from curve relating T and concn. Ex- 
press chlorophyll values as mg/g tissue, or in other convenient 
manner. 

Calibrate photoelec. colorimeter as follows: Ext sample of 
fresh, green leaf material with 85% acetone, filter, wash res- 
idue, and dil. ext to vol. as above. Make series of dilns of ext 
and measure T of original and of each of dild solns with in- 
strument in same manner as when chlorophyll prepn is being 
used as calibration std. Transfer aliquot of original ext to ether 
and evaluate total chlorophyll spectrophtric as in 942.04C(b) 
and (c). From value thus obtained, calc. chlorophyll content 
of original ext and that of each of dild solns, and construct 
curve relating concn of chlorophyll with T ot A. 

Refs.: Ind. Eng. Chem. Anal. Ed. 12, 148(1940); 15, 524 
(1943). 

CAS- 1406-65-1 (chlorophyll) 



PIGMENTS 

940.03 Chlorophyll in Plants 

Photoelectric Colorimetric Method for 
Total Chlorophyll Only 

Final Action 

A. Apparatus 

(a) Mortar and pestle. — Deep glass mortar ca 10 cm id with 
well-defined lip. 

(b) Photoelectric colorimeter. — Calibrate for chlorophyll, 
using plant ext as in 940. 03C and light filters with max. T 
near 660 nm. (Combination of Kopp Glass Works filters K2408 
and K3965 is suitable.) 

(c) Wash bottles. — Type fitted with rubber bulb, permitting 
operation with one hand. 

(d) High-speed blender. — Waring Blendor, or equiv. 

B. Reagents 

(a) Acetone . — (1) Undild acetone and (2) 85% aq. soln by 
vol. Com. acetone, tech. grade, is satisfactory. 

(b) Quartz sand. — Acid- washed and dried. 

C. Determination 

(Caution: See safety notes on blenders, toxic solvents, and 
acetone.) 

Select field material carefully to ensure representative sam- 
ple. Remove representative portion from field sample, and if 
fresh, cut finely with hand shears and mix as thoroly as pos- 
sible. Grind dried material in mill and mix thoroly. 



942.04 Chlorophyll in Plants 

Spectrophotometry Method for Total Chlorophyll 
and the a and b Components 

Final Action 

A. Apparatus 

Use app. in 940. 03 A (except photoelec. colorimeter), plus 
following: 

(a) Scrubbing tubes for washing ether solns. — Open tubes 
ca 20 mm diam. to one end of each of which is sealed tube 
of smaller diam. drawn to fine jet at lower end. 

(b) Spectrophotometer . — Capable of isolating spectral re- 
gion of ca 3 nm near 660 nm with negligible stray radiation. 
Tubulated cells with tightly fitting glass stoppers are recom- 
mended for work with ether. 

B. Reagents 

Those listed in 940. 03B and following: 
Ether. — Com. grade is satisfactory without further purifi- 
cation. 

C. Determination 

(Wash glassware with coned Na 3 P0 4 soln to remove traces of 
acid that may decompose chlorophyll.) 

(a) Extraction of chlorophyll from tissue. — Select and prep, 
sample as in 940. 03C. Disintegrate weighed portion (2-10 g, 
depending on chlorophyll content) of fresh plant tissue in blender 
cup that contains ca 0.1 g CaC0 3 , or by use of mortar as in 
940. 03C. After tissue is thoroly disintegrated, filter ext thru 
buchner fitted with quant, paper. Wash residue with 85% ace- 



AOAC Official Methods of Analysis (1990) 



Pigments 



63 



tone, 940.03B(a), and if necessary, use little ether to remove 
last traces of pigment. If extn is incomplete, return residue and 
paper to blender container with more 85% acetone and repeat 
extn. Filter and wash, as before, into flask contg first filtrate. 
Transfer filtrate to vol. flask of appropriate size and dil. to 
vol. with 85% acetone. 

Pipet aliquot of 25-50 mL into separator contg ca 50 mL 
ether. Add H 2 carefully until it is apparent that all fat-sol. 
pigments have entered ether layer. Drain and discard H 2 layer. 
Place separator contg ether soln in upper rack of support. Add 
ca 100 mL H 2 to second separator placed in rack below first. 
Set scrubbing tube in place, and let ether soln run thru it to 
bottom of lower separator and rise in small droplets thru the 
H 2 0. When all soln has left upper separator, rinse it and scrub- 
bing tube with little ether added from medicine dropper. Place 
scrubbing tube in upper separator and exchange its place in 
support with separator now contg ether soln. Drain and discard 
H 2 in upper separator, add similar portion of fresh H 2 to 
lower separator, and repeat washing process. Continue wash- 
ing ether soln until all acetone is removed (5-10 washings). 
Then transfer ether soln to 100 mL vol. flask, dil. to vol., and 
mix. 

(b) Spectrophotometric measurements . — Add ca teaspoon- 
ful (ca 5 mL) anhyd. Na 2 S0 4 to 60 mL reagent bottle, and fill 
it with ether soln of pigment. When this soln is optically clear, 
pipet aliquot into another dry bottle and dil. with enough dry 
ether to give A value of 0.2-0.8 at wavelength to be used. 
(Most favorable value is near 0.6 at 660 nm, since such soln 
yields satisfactory value at 642.5 nm.) 

Fill 2 clean g-s absorption cells with dry ether from pipet 
and polish outside surfaces of each, first with cotton wet with 
alcohol and then with dry cotton. Place cells in instrument, 
and det. whether each gives same galvanometer deflection. If 
not, clean again or select cells that do, and do this daily. Empty 
one cell, fill it with the dried ether soln, and place in instru- 
ment. Adjust entrance and exit slits until spectral region iso- 
lated is 3-4 nm at 660.0 nm. 

Det. whether instrument is in proper adjustment for wave- 
length by taking A readings thru soln against solv. at 1 nm 
intervals from 658 to 665 nm. Highest value should be at 660.0 
nm; if not, adjust instrument until it is, or make 660.0 nm 
readings at wavelength setting that gave highest A. With grat- 
ing instrument, apply same correction at 642.5 nm; however, 
with prism instrument, correction at 642.5 nm must be ob- 
tained from wavelength calibration curve for particular instru- 
ment in use. Calibrate instrument for wavelength in this way 
often enough to ensure that it remains in proper adjustment. 
Det. A at 660.0 and 642.5 nm (or corrected settings) for each 
unknown soln. 

(c) Calculation of chlorophyll concentration. — Calc. total 
chlorophyll and each of a and b components (mg/L) as fol- 
lows: 

(1) Total chlorophyll - 7.12A 660 . + 16.8A 6425 

(2) Chlorophyll a - 9.93 A 660 . - 0.777 A M2 _ 5 

(3) Chlorophyll b = 17.6 A 642 . 5 - 2.81 A 660 . 
D. Supplementary information 

Factors involved in spectrophtric analysis of chlorophyll 
system have been discussed in detail (Plant Physiol. 17, 198 
(1942)). These authors used Beer's law in form: 

c = Qogiolo/0/a X t [== A/a X t\ 

where / is intensity of light transmitted by solv. -filled cell; / 
is intensity of light transmitted by sol n-fi lied cell; c is concn 
of chlorophyll (g/L); a is absorptivity; / is thickness of soln 
layer in cm; and A is absorbance. 



Table 942.04 Absorption Constants Used in Analysis 
(after Cornar and Zscheile) 



Wavelength, 


Absorptivities (for Ether Solns) 


nm 


Chlorophyll a 


Chlorophyll b 


660.0 


102 


4.50 


642.5 


16.3 


57.5 


600.0 


9.95 


9.95 


581.0 


8.05 


8.05 


568.0 


7.11 


7.11 


613.0 


15.6 


8.05 


589.0 


5.90 


10.3 



Since, at given wavelength, observed A value of soln having 
2 components represents sum of A values of each of compo- 
nents, following equation holds in case of chlorophylls a and 
b at given wavelength: 

(4) ^observed ~ A a + A b 

If 1 cm cell is used, this equation may be expressed as: 



(5) 



= a a c a + a b c b 



Concns of chlorophylls a and b in given ether soln can now 
be calcd by equation (5) as follows: 

(a) Det. A for soln at 2 different wavelengths (660.0 and 
642.5 nm have been found advantageous for this purpose). 

(b) From Table 942.04, select proper absorptivities corre- 
sponding to wavelengths used. 

(c) Substitute observed A value and absorptivities in equa- 
tion (5) for each of the 2 wavelengths used as illustrated for 
660.0 and 642.5 nm in equations (6) and (7). Solve these 2 
equations simultaneously for 2 unknowns, the concns of chlo- 
rophylls a and b. 



(6) 
(7) 



= 102 c a + 4.50 c h 



16.3 c a + 57.5 c h 



Equations (1), (2), and (3) were derived this way. 

Criterion for accuracy of chlorophyll values detd by spec- 
trophtric method is agreement between analytical results detd 
from measurements at different wavelengths. Measurements at 
660.0 and 642.5 nm are convenient for routine analysis (Plant 
Physiol. 17, 198 (1942)); however, readings may be made at 
other wavelengths to check these values. Absorptivities for 
chlorophylls a and b in ether soln that may be used for this 
purpose are presented in Table 942.04. 

These values may be used for calcns as follows: 

(a) Values for total chlorophyll and % composition may be 
calcd from A at 660.0 and 642.5 nm as described. 

(b) Check values for total chlorophyll may be calcd from 
A at intersection points 600.0, 581.0, and 568.0 nm. 

(c) Check values for % composition may be calcd from A 
for each of points 613.0 and 589.0 nm in combination with 
value of total concn obtained from (a) or (b). 

Refs.: Ind. Eng. Chem. Anal. Ed. 14, 877(1942). Plant Phys- 
iol. 17, 198(1942). 

CAS- 1406-65-1 (chlorophyll) 



955.10 



See 941.15. 



Carotenes in Plants 
Final Action 



64 



Plants 



AOAC Official Methods of Analysis (1990) 



TOBACCO 

966.02 Moisture in Tobacco 

Gravimetric Method 

First Action 1966 
Final Action 1968 

A. Apparatus 

(a) Drying oven. — Forced-draft, regulated to 99.5±0.5°. 
Suggested dimensions: 19 x 19 x 19" (48 cm). Approx. oven 
settings: fresh air intake vent Vs open; air control damper 1 / 4 
open; air exhaust vent } / 3 open. 

(b) Moisture dish. — Al, diam. 45-65 mm, depth 20-45 mm, 
with tight-fitting cover. 

B. Determination 

Accurately weigh ca 5 g sample (ground to pass <1 mm 
screen) into weighed moisture dish and place uncovered dish 
in oven. 

Do not exceed 1 sample/ 10 sq in. (650 sq cm) shelf space, 
and use only 1 shelf. Dry 3 hr at 99. 5 ±0.5°. Remove from 
oven, cover, and cool in desiccator to room temp, (ca 30 min). 
Reweigh to nearest 1 mg and calc. % moisture. 

Ref.: JAOAC 49, 525(1966). 



963.05 Chiorides in Tobacco 

Potentiometric Method 

First Action 1963 
Final Action 1964 

A. Reagents 

Silver nitrate std soln. — 0.1 N. Stdze against KC1 as in detn. 

B. Apparatus 

(a) pH meter. — Leeds and Northrup, Sumneytown Pike, N 
Wales, PA 19454, Beckman Instruments, or equiv., equipped 
with Ag and glass electrodes. 

(b) Buret. — 10 mL, graduated in 0.05 or 0.02 mL, pref- 
erably reservoir-type. 

C. Determination 

Accurately weigh ca 2 g sample, ground to pass No. 40 
sieve, into 250 mL electrolytic beaker. Add 100 mL H 2 0, small 
amt at first to thoroly wet sample; then remainder. Let stand 
>5 min at room temp. , stirring intermittently. Pipet 5 mL HN0 3 
(1+9) into mixt. and insert clean electrodes. Start mag. stirrer 
and continue stirring thruout titrn at rate that produces vigor- 
ous agitation without spattering. Titr. with std 0.1 N AgN0 3 
soln to potential previously established as equivalence point. 
Det. equivalence point potential graphically by making several 
titrns on one or more tobacco samples. Recheck occasionally, 
and redet. when either electrode is replaced. Record vol. of 
titrant and calc: 

% CI = mL AgN0 3 x normality x 3.5453/g sample 

Ref.: JAOAC 46, 415(1963). 



959.04 Nitrogen in Tobacco 

Kjeldahl Method for Samples 
Containing Nitrates 

Final Action 1964 

(For nitrate-free samples, omit salicylic acid and thiosulfate 
treatment.) 



A. Reagents 

See 920.02A and the following: 

(a) Sodium hydroxide -thiosulfate soln. — Dissolve 500 g 
NaOH pellets and 40 g Na 2 S 2 3 .5H 2 in H 2 and dil. to 1 L. 

(b) Indicators. — (7) Dissolve 1 g Me red in 200 mL al- 
cohol; or (2) prep, mixed indicator by dissolving 0.8 g Me red 
and 0.2 g methylene blue in 500 mL alcohol. 

B. Apparatus 
See 920.02B. 

C. Determination 

Place weighed sample (1-2 g) in digestion flask. Add vol. 
H 2 S0 4 (contg 2 g salicylic acid/40 mL) corresponding to wt 
sample (35 mL for 1 g, 40 mL for 2 g for N0 3 -contg samples; 
20 and 25 mL, resp., for N0 3 -free samples). Shake until tho- 
roly mixed; let stand >30 min with occasional shaking; then 
add 5 g Na 2 S 2 3 .5H 2 0. Shake, let stand 5 min, and heat care- 
fully until frothing ceases. Turn off heat, add 0.7 g HgO (or 
metallic Hg) and 15 g K 2 S0 4 , and boil briskly 1-1.5 hr after 
soln clears. 

Cool, add ca 200 mL H 2 0, cool to ca room temp. , and add 
few Zn granules. Tilt flask and carefully add 50 mL NaOH- 
thiosulfate soln without agitation. Immediately connect flask 
to distn bulb on condenser whose tip is immersed in 50 mL 
std 0.1 N acid in receiving flask. Then rotate digestion flask 
carefully to mix contents. Heat until >150 mL distillate col- 
lects, and titr. excess acid with std base, using Me red or mixed 
indicator. Correct for blank detn on reagents. 

Ref.: JAOAC 42, 302(1959). 

CAS-7727-37-9 (nitrogen) 



966.03 Potassium in Tobacco 

Flame Photometric Method 

First Action 1966 
Final Action 1968 

A. Reagents 

(a) Potassium std so Ins. — (I) Stock soln. — 1000 ppm K. 
See 956.01A(a). (2) Working solns.— Place 0, 5, 10, 15, 20, 
25, and 30 mL stock soln in seven 1 L vol. flasks, add 40 mL 
3 N HC1 to each, and dil. to vol. with H 2 0. 

(b) Diatomaceous earth. — Celite 545, acid-washed. 

B. Apparatus 

(a) Flame photometer. —Natural gas-air fuel, or equiv., ad- 
equate for K analysis. 

(b) Chromatographic tube. — 20 x 150 mm with coarse 
fritted disk. 

C. Preparation of Sample Solution 

Accurately weigh ca 0.5 g tobacco dust into ca 40 mL 
weighing dish. Add ca 1 g Celite and mix intimately with spat- 
ula. Transfer quant, thru powder funnel into chromatgc tube. 
Add addnl Celite thru funnel into tube until 2.5 cm layer ac- 
cumulates on top of sample-Celite mixt. Compact sample and 
Celite by tapping tip of tube on table top, and insert tip of tube 
into neck of 1 L vol. flask. Add 40 mL 3N HCl into tube by 
pipet or dispenser, washing down sides, and let elute into vol. 
flask. When liq. level reaches top of Celite, add 25 mL H 2 
and let elute. Add second 25 mL portion of H 2 0, let elute by 
gravity, or force thru rapidly with compressed air. Rinse tip 
of tube into vol, flask, dil. to vol. with H 2 0, and mix well. 



AOAC Official Methods of Analysis (1990) 



Tobacco 



65 



D. Determination 

Det. % T for sample eluate and K stds as specified in in- 
struction manual of instrument. See also 956. 01D. 

Prep, calibration curve and det. ppm K of sample from curve. 

% K = ppm KxO.l/g sample 
% K 2 - ppm K x 0.1205/g sample 

Ref.: JAOAC 49, 521(1966). 

CAS-7440-09-7 (potassium) 



971.02 Glycerol, Propylene Glycol, 

and Triethylene Glycol in Cased Cigarette Cut Filler 
and Ground Tobacco 
Gas Chromatographic Method 

First Action 1971 
Final Action 1985 

(Caution: See safety notes on pipets and methanol.) 

A. Apparatus 

(a) Gas chromato graph. — With programmed temp, oven 
and W hot wire detector; F&M Model 720 (current models 
5700 series; Hewlett-Packard Co.), or equiv. Conditions: De- 
tector bridge 140 ma; temps (°): injection 265, detector 280, 
column 90-240 at 15°/min; He 60 mL/min adjusted, if nec- 
essary, to facilitate sepns; attenuation 4, adjusted according to 
sensitivity to yield peaks of sufficient size for accurate mea- 
surement (use same attenuation for all stds and samples); chart 
speed, 12"/hr. 

(b) Column. — 42 (105 cm) x 3 / 16 " Cu tubing packed with 
5% Carbowax 20M-terephthalic acid (TPA) on 60-80 mesh 
Chromosorb G AW-DMCS (Hewlett-Packard Co., No. 8501- 
6223 or Applied Science No. 04388). Prep, packing by plac- 
ing 30.0 g Chromosorb in 500 mL 1" r-b flask. Add soln of 
1.50 g Carbowax 20M-TPA in 150 mL CHC1 3 , and slurry. 
Remove CHC1 3 under vac. in rotary evaporator and air dry 
overnight at room temp. Condition new column 2 hr at 240°; 
then inject three 30 jjlL samples tobacco ext before analyzing 
samples. Recondition columns removed from app. before use. 

B. Reagents 

(a) Extracting soln. — Dil. 20.0 mL 1 ,3-butylene glycol stock 
std soln, (b), to 2 L with anhyd. MeOH. 

(b) 1,3-Butylene glycol stock std soln. — Accurately weigh 
20.00 g USP 1,3-butanediol into 100 mL vol. flask and dil. 
to vol. with anhyd. MeOH. 

(c) Glycerol stock std soln. — Accurately weigh 10.00 g USP 
glycerol into 100 mL vol. flask and dil. to vol. with extg soln. 

(d) Propylene glycol stock std soln. — Accurately weigh 5.00 
g USP propylene glycol into 100 mL vol. flask and dil. to vol. 
with extg soln. 

(e) Triethylene glycol stock std soln. — Accurately weigh 5.00 
g triethylene glycol into 100 mL vol. flask and dil. to vol. 
with extg soln. 

(f) Humectant std solns. — Into each of four 100 mL vol. 
flasks, pipet 1.0, 2.0, 3.0, and 4.0 mL, resp., glycerol, pro- 
pylene glycol, and triethylene glycol stock std solns. Dil. to 
vol. with extg soln. Each soln contains (in mg/100 mL): 





Propylene 




Triethylene 


Soln 


Glycol 


Glycerol 


Glycol 


1 


50 


100 


50 


2 


100 


200 


100 


3 


150 


300 


150 


4 


200 


400 


200 



C. Extraction 

Place 10.00 g sample in 250 mL ¥ erlenmeyer. Pipet 100 
mL extg soln into flask and stopper. Shake mech. 1 hr and let 
settle few min until supernate is clear. Alternatively, shake 
mech. 30 min and let stand overnight. 

D. Determination 

Prime column by injecting two 30 jjlL aliquots supernate ext. 
Then alternately inject 30 (jlL supernate exts and a humectant 
std soln until all samples and stds have been run, repeating 
ext injections, if necessary. (Sequence is exti, ext,, std, , ext], 
std 2 , ext 2 , std 3 , ext 3 , std 4 , ext 4 , std 1? ext 5 , std 2 , etc. if <4 exts 
are available, distribute ext injections among those available 
so that sequence thru std 4 is run.) Det. peak hts and calc. ratios 
of propylene glycol, glycerol, and triethylene glycol to butyl- 
ene glycol for each std and sample soln. Plot peak ht ratios 
against polyol concn for std solns and construct std curve for 
each humectant. Det. concn in mg/100 mL for propylene gly- 
col, glycerol, and triethylene glycol in sample soln from resp. 
std curves. 

% Humectant - (mg/100 mL) X 0.01 

Ref.: JAOAC 54, 560(1971). 

CAS-56-81-5 (glycerol) 

CAS-57-55-6 (propylene glycol) 
CAS-1 12-27-6 (triethylene glycol) 



960.07 Alkaloids (Total 

As Nicotine) in Tobacco 
Distillation Method 

First Action 1960 
Final Action 1964 

A. Apparatus 

(a) Distillation apparatus. — 500 mL Kjeldahl flask fitted 
with inlet tube for steam, trap bulb, and condenser; Griffith 
still (Tobacco Sci. 1, 130(1957), available from Lab Glass, 
Inc., PO Box 610, Vineland, NJ 08360); or other suitable steam 
distn app. 

(b) Spectrophotometer. — Beckman Instruments Model DU, 
24, or 25 (replacement model DU-64), or other instrument ca- 
pable of accurately measuring A in 200-300 nm range, equipped 
with 1 cm quartz cells. 

B. Reagents 

(a) Alkali-salt soln.— Dissolve 300 g NaOH in 700 mL H 2 
and sat. with NaCl. 

(b) Silicotungstic acid soln (for gravimetric determina- 
tion).— Dissolve 120 g Si0 2 .12W0 3 .26H 2 in H 2 and dil. 
to 1 L. (Soln should be clear and free from green color.) 

C. Standardization 

(Caution: Nicotine is very toxic. Avoid contact with skin and 
eyes. See safety notes on distillation and vacuum.) 

Purify best grade of nicotine com. available by successive 
vac. distns until center cuts from 2 successive distns have same 
a at 259 nm (ca 34.3). Accurately weigh ca 0.2 g purified 
nicotine; dissolve in and dil. to 1 L with ca 0.05 N HC1. Dil. 
10 mL aliquot of this soln to 100 mL with ca 0.05 W HC1. 
Det. A at 259 nm and calc. a = A/(c x b), where c is concn 
of nicotine in g/L and b is cell length in cm. 

D. Distillation 

Accurately weigh 2-5 g tobacco sample and transfer to distn 
flask or app. (If final detn of nicotine is gravimetric, use sam- 



66 



Plants 



AOAC Official Methods of Analysis (1990) 



pie contg ^0.1 g alkaloids; if spectrophtric, use >2 g sample.) 
(If Griffith still is used, use 0.05-0.2 g sample.) Place 25 mL 
HC1 (1+4) in receiver (1 L vol. flask is desirable) and place 
receiver so that condenser tube dips into acid. (With Griffith 
still, use 10 mL HC1 (1+4) in 250 mL vol. flask.) Add 50 mL 
alkali-salt soln to distn flask so that sample is rinsed into bot- 
tom of flask. (With Griffith still, use 5 mL alkali-salt soln.) 
If large vol. of liq. is required for proper function of still, add 
more alkali-salt soln; do not dil. Connect flask to app. im- 
mediately and steam distil with as rapid current of steam as 
can be condensed efficiently. Effluent condensate should not 
be above room temp. Apply heat to distn flask from burner, 
mantle, or other heat source to keep vol. in flask approx. const. 
Collect ca 900 mL condensate (or distil addnl 100 mL after 
condensate shows no nicotine by silicotungstic acid test). (With 
Griffith still, collect 225 mL.) Dil. distillate to vol. 

E. Determination 

(a) Spectrophotometries — Dil. aliquots of distillate (if nec- 
essary) with 0.05 7V HC1 so that A at 259 nm is 0.5-0.8 and 
read A at 236, 259, and 282 nm. Calc. corrected A' 259 = 1.059 
x [observed A 259 - Vs (^ 236 + ^282)] a f ter correcting all ob- 
served A values to original distillate vol. basis. Concn, c, of 
alkaloids as nicotine in g/L is given by c ~ A' 259 /(a X /?), 
where a is absorptivity at 259 nm, and b is ceil length in cm. 

% alkaloid (as nicotine) = c 

X vol. distillate (L) X 100/g sample 

(b) Gravimetric. — Det. alkaloids in distillate as in 920.35B, 
but double amt of silicotungstic acid specified, i.e., 2 mL/ 
each 10 mg alkaloids expected. 

Ref.: JAOAC 43, 524(1960). 

CAS-55-11-5 (nicotine) 



960.08 Alkaloids (Total As Nicotine) 

in Tobacco 
Cundiff-Markunas Method 

First Action 1960 
Final Action 1964 

(Total alkaloids (as nicotine), tertiary alkaloids (as nicotine), 
and secondary alkaloids (as nornicotine)) 

A. Reagents 

(a) Benzene-chloroform soln.— Mix equal parts by vol. of 
benzene and CHCl 3 and sat. with H 2 0. 

(b) Sodium hydroxide soln. — 36%. Dissolve 500 g NaOH 
in H 2 and dil. to 1 L. 

(c) Dilute acetic acid. — 5%. Dil. 50 mL HOAc to 1 L with 
H 2 0. 

(d) Crystal violet indicator. — Dissolve 0.5 g crystal violet 
in 100 mL HOAc. 

(e) Perchloric acid std soln. — 0.025 7V. Add 4.7 mL 72% 
HCIO4 to freshly opened 5 lb bottle HOAc and mix. {Caution: 
See safety notes on acetic acid, acetic anhydride, and perchlo- 
ric acid.) Stdze as follows: Accurately weigh 0.1 g KH phthal- 
ate (NIST) into 125 mL erlenmeyer, add 50 mL HOAc, and 
heat to dissolve. Cool, add 2 drops indicator, and titr. to blue- 
green end point. Perform blank titrn on 50 mL HOAc and 2 
drops indicator soln, and correct vol. of titrant. 

N = wt KH phthalate x 4.896/mL HC10 4 soln 

B. Determination 

Accurately weigh 2.5 g finely ground tobacco into 250 mL 
erlenmeyer. Add 15 mL 5% HOAc and swirl until tobacco is 



thoroly wetted. Pipet 100 mL benzene-CHCl 3 soln into flask, 
and then 10 mL 36% NaOH soln. Stopper flask tightly and 
shake 20 min, using wrist-action shaker. Add 4.5-5 g (2 tea- 
spoonfuls) Filter-Cel, mix, and filter most of benzene layer 
thru Whatman No. 2 paper into second flask. If filtrate has 
any turbidity, add 2-2.5 g (1 teaspoonful) addnl Filter-Cel and 
ref i Iter thru Whatman No. 2 paper. Filtrate must be clear. 

Pipet 25 mL aliquots of filtrate into each of two 125 mL 
erlenmeyers. Pass stream of air over surface of soln in first 
flask 5 min, add 2 drops indicator, and titr. to green end point 
with 0.025 TV HCIO4. Add 1.0 mL Ac 2 to second flask and 
let stand >15 min. Add 25 mL HOAc and 2 drops indicator, 
and titr. to blue-green end point with 0.025 N HCIO4. Take 
first appearance of blue-green thruout soln as end point. For 
each series of analyses perform blank titrn s and correct re- 
spective vols of titrant. 

Calc. % alkaloids as follows: % total alkaloids (as nicotine) 
= Vj X TV X 32.45/wt sample; % tertiary alkaloids (as nico- 
tine) = (2V 2 ~ Vi) X N X 32.45/wt sample; % secondary 
alkaloids (as nornicotine) - 2{V X - V 2 ) X 29.64/wt sample; 
where V x = vol. titrant for nonacetylated aliquot; V 2 — vol. 
titrant for acetylated aliquot; and N - normality HCIO4. 

Ref.: JAOAC 43, 524(1960). 

CAS-55-11-5 (nicotine) 

CAS-494-97-3 (nornicotine) 



979.01 Nicotine on Cambridge Filter Pads 

Gas Chromatographic Method 

First Action 1979 
Final Action 1984 

A. Apparatus and Reagents 

(a) Gas chromato graph. — With flame ionization detector, 
heated injection port, and thermostated column oven. Follow- 
ing conditions have been found satisfactory: Column, 1.8 m 
(6') x Vs" stainless steel; packing, 2% KOH and 10% Car- 
bowax 20M (based on final packing wt) on 45-60 mesh cal- 
cined diat. earth (such as Chromosorb W, or equiv.), resieved 
before use to mesh range to remove fines and lumps; temps 
(°): column 165, detector and injection port 200-250; carrier 
gas flow, ca 40 mL/min. Adjust H and air Hows for max. 
sensitivity and stability. Under these conditions, column should 
have ht equiv. to theoretical plate (HETP) <1 mm and reso- 
lution of >2, calcd with nicotine and anethole. 

(b) Measuring system. — Measure peak areas with elec- 
tronic integrator or other system with resolution of 2: 1 count/ 
mv-sec. 

(c) Mechanical shaker. — Capable of extg ^99% nicotine. 
Burreil Wrist- Action shaker has been found satisfactory. 

(d) Extracting soln. — 2-Propanol contg 1 mg anethole/mL 
as internal std for nicotine. If H 2 is also to be detd, add 20 
mg EtOH/mL 2-propanol as addnl internal std. 

(e) Nicotine std solns. — (/) Stock soln. — Weigh 2.500 g 
nicotine, 960.07C, or equiv. amt of nicotine salt. Transfer quant. 
into 100 mL vol. flask, and dil. to vol. with extg soln. (2) 
Working std solns. — Pipet 1, 2, 3, 4, and 5 mL stock soln 
into five 100 mL vol. flasks, and dil. to vol. with extg soln 
(0.25, 0.50, 0.75, 1.00, and 1.25 mg nicotine/mL).(CawfK>/i; 
See precaution in 960. 07C.) 

B. Extraction 

Place Cambridge filter material in flask or serum bottle ac- 
commodated by shaker used, add 10.00 mL extg soln, stopper, 
and shake until >99% of nicotine is extd (usually ca 15 min). 



AOAC Official Methods of Analysis (1990) 



Tobacco 



67 



C. Standardization 

Prime column with aliquots of 1.25 mg/mL std soln. Let 
baseline stabilize, inject 1 |iL each std soln in succession, and 
repeat sequence 3 times. Det. area ratio (nicotine:anethole) for 
each injection, and calc. slope and intercept of response curve, 
preferably by method of least squares (See Definition of Terms 
and Explanatory Notes). Correlation coefficient should be >0.99 
and intercept <0.05 mg/mL. 

D. Determination 

Prime column with aliquots of ext, 979.01B. Let baseline 
stabilize, and inject 1 jjlL of each sample soln. Calc. nicotine 
concn in soln (C, mg/mL) = mx + b, where m = slope of 
stdzn curve, b = intercept, and x — area ratio of nicotine to 
anethole. 

Nicotine yield /cigarette = (C X 10.00)/(No. cigarettes /pad) 

Ref.: J AOAC 62, 229(1979). 

CAS-55-11-5 (nicotine) 



968.02 Menthol in Cigarette Filler 

Colorimetric Method 

First Action 1968 
Final Action 1970 

A. Apparatus and Reagents 

(a) Distillation apparatus .—See Fig. 968.02. 

(b) Spectrophotometer. — With matched cells; capable of 
measuring A at 550 nm. 

(c) Menthol std. soln. — 1 mg/mL. Accurately weigh 100 
mg USP /-menthol into 100 mL vol. flask, add alcohol to dis- 
solve, and dil. to vol. with alcohol. 

(d) DMAB color reagent. — Dissolve 0.5 g p-dimethylami- 
nobenzaldehyde (Sigma Chemical Co.) in 100 mL H 2 S0 4 
(1.6+1). 

B. Preparation of Calibration Curve 

Prep. dil. stds by pipeting aliquots contg 0, 3, 4, 6, 8, and 
10 mg menthol into 100 mL vol. flasks and dilg to vol. with 
alcohol (1 + 1). Pipet 1 mL each dil. std into 10 mL test tube, 
add 5 mL color reagent, mix, and place in boiling H 2 bath 
exactly 2 min. Cool in tap H 2 0, and within 15 min det. A at 
550 nm against std. Prep, calibration curve by plotting A 
against menthol concn (mg/100 mL). 

C. Determination 

Accurately weigh 2.00-2.15 g cigaret filler and transfer to 
distn flask, A. Add 80 ml H 2 and few boiling stones, con- 
nect flask to condenser with tube, B, attach adapter, C, to 
condenser, and immerse tip in 20 mL alcohol in 100 mL vol. 
receiving flask. 

Gently heat distn flask until distn begins; then increase heat 
and lower receiving flask, D, so tip of adapter is no longer 
immersed. Distil until 20 mL distillate collects. Disconnect 
condenser from tube, and wash down condenser with alcohol. 
Remove receiving flask, dil. distillate to ca 70 mL with al- 
cohol, and add H 2 almost to vol. Mix, add alcohol to vol., 
and mix again. 

Pipet 1 mL distillate into 10 mL test tube, add 5 mL color 
reagent, mix, and place in boiling H 2 bath exactly 2 min. 
Cool in tap H 2 0, and within 15 min det. A at 550 nm, using 
"color" soln from nonmentholated tobacco carried thru detn 
as blank. (If nonmentholated sample corresponding to men- 
tholated sample is not available, use reagent blank.) Use non- 
mentholated tobacco blank within 15 min after color devel- 
opment step. Fresh nonmentholated tobacco blank soln may 



be required during multiple sample runs. Det. mg menthol from 
calibration curve. 

% Menthol = mg menthol /(g original sample x 10) 

Ref.: JAOAC51, 650(1968). 

CAS-1490-04-6 (menthol) 



968.03 Menthol in Cigarette Filler 

Gas Chromatographic Method 
Final Action 1970 

A. Apparatus and Reagents 

(a) Gas chromatograph. — Equipped with flame ionization 
detector and thermostated injection port and column oven. Use 
following conditions for analysis: Column, 1.5 m (5') X ] /s" 
od stainless steel packed with 10% (w/w) silicone oil DC-550 
on 60-80 mesh Chromosorb W; temps (°): column 150, de- 
tector 150, injection port 175; N carrier gas flow ca 35 mL/ 
min. Adjust H and air flows for max. sensitivity and reason- 
able stability. 

(b) Mechanical shaker. — Wrist action. 

(c) Menthol-anethole std. soln. — 0.250 mg menthol and 0.50 
mg anethole/mL. Weigh exactly 0.5000 g tech. grade aneth- 
ole and wash into 1 L vol. flask with 200 mL alcohol. Transfer 
0.2500 g USP /-menthol to the vol. flask with enough alcohol 
to bring to vol. Store soln in dark g-s bottle. Do not use >6 
weeks. 

(d) Extracting soln. — 0.50 mg anethole/mL. Dissolve 1 .000 
g anethole in alcohol in 2 L vol. flask, dil. to vol. with alcohol, 
and store in dark. 

B. Determination of Ratio Factor 

Weigh ca 3 g nonmentholated control filler, contg all usual 
humectants but no menthol or anethole, into 125 mL rubber- 
stoppered flask. Pipet 50 mL std menthol-anethole soln into 
flask, stopper, and shake 1 hron mech. shaker. Let solids set- 
tle 15 min and chromatograph 2 pJL aliquot of supernate. Re- 
peat twice more to obtain total of 3 replicates of std chro- 
matogram. For quant, results, inject both std and unknown 
samples by inserting 2" (5 cm) needle to hilt, injecting 2 (jlL 



MM QD. TUBING 
24/40 
500 ML 




100 ML 



FIG. 968.02— Distillation apparatus; see 968.02C for 
explanation of symbols 



68 



Plants 



AOAC Official Methods of Analysis (1990) 



rapidly, and withdrawing needle at once. (Menthol elutes in 
ca 3 min, anethole in ca 5 min.) After ca 10 min, all other 
compds are eluted and new injection can be made. 

Draw baselines under menthol and anethole peaks and mea- 
sure peak hts in mm. Using mean peak ht of menthol and 
anethole from 3 std chromatograms, calc. std ratio factor of 
menthol to anethole as follows: 

Std ratio factor = peak ht for menthol (0.25 mg/mL)/ 

peak ht for anethole (0.50 mg/mL) 

C. Determination 

Accurately weigh 8-8.5 g mentholated cigarette filler and 
place in 250 mL rubber-stoppered erlenmeyer. Pipet 100 mL 



extg soln into flask, stopper, and mech. shake 2 hr. Let solids 
settle 15 min and chromatograph 2 |xL aliquot of supernate. 
Draw baselines under menthol and anethole peaks and measure 
peak hts in mm. Calc. ratio factor of unknown menthol as 
follows: 

Ratio factor for unknown = peak ht for unknown menthol/ 
peak ht for anethole (0.50 mg/mL) 

% Menthol = (unknown ratio factor x 0.25 x 10)/ 

(std ratio factor x g sample) 

Ref.: JAOAC 51, 650(1968). 

CAS- 1490-04-6 (menthol) 



4 a Animal Feed 

Joel WL Padmore, Associate Chapter Editor 

North Carolina Department of Agriculture 



965.16 Sampling of Animal Feed 

Procedure 

Use slotted single or double tube, or slotted tube and rod, 
all with pointed ends. 

Take >500 g sample, 1 kg preferred, as follows: Lay bag 
horizontally and remove core diagonally from end to end. Det. 
number of cores as follows: From lots of 1-10 bags, sample 
all bags; from lot of ^11, sample 10 bags. Take 1 core from 
each bag sampled, except that for lots of 1-4 bags take enough 
diagonal cores from each bag to total >5 cores. For bulk feeds 
draw >10 cores from different regions; in sampling small con- 
tainers (<10 lb) 1 package is enough. Reduce composite sam- 
ple to amt required, preferably by riffling, or by mixing 
thoroly on clean oil-cloth or paper and quartering. Place sam- 
ple in air-tight container. 

A sample from less than these numbers of bags may be de- 
clared an official sample if guarantor agrees. For samples that 
cannot be representatively taken with probe described, use other 
sampling means. 



950.02 



Animal Feed 

Preparation of Sample 

Final Action 



Grind sample to pass sieve with circular openings 1 mm 
(V25") diam. and mix thoroly. If sample cannot be ground, re- 
duce to as fine condition as possible. Do not grind molasses 
feeds. 

Refs.: JAOAC 33, 424(1950); 41, 223(1958); 48, 658(1965); 
51, 467(1968). 



0.01 mL. Clean tube and condenser with chromic acid clean- 
ing mixt., rinse thoroly with H 2 0, then alcohol, and dry in 
oven to prevent undue amt H 2 from adhering to inner sur- 
faces during detn. 

B. Determination 

If sample is likely to bump, add dry sand to cover bottom 
of flask. Add enough toluene to cover sample completely (ca 
75 mL). Weigh and introduce enough sample into toluene to 
give 2-5 mL H 2 and connect app. Fill receiving tube with 
toluene, pouring it thru top of condenser. Bring to boil and 
distil slowly, ca 2 drops /sec, until most of the H 2 passes 
over; then increase rate of distn to ca 4 drops/sec. 

When all H 2 is apparently over, wash down condenser by 
pouring toluene in at top, continuing distn short time to see 
whether any more H 2 distils over; if it does, repeat washing- 
down process, if any H 2 remains in condenser, remove by 
brushing down with tube brush attached to Cu wire and satd 
with toluene, washing down condenser at same time. (Entire 
process is usually completed within 1 hr.) Let receiving tube 
come to room temp. If any drops adhere to sides of tube, force 
them down, using Cu wire with end wrapped with rubber band. 
Read vol. H 2 and calc. to %. 

Refs.: JAOAC 8, 295(1925); 9, 30(1926). 



920.36* Moisture in Animal Feed 

Drying without Heat over 
Sulfuric Acid 

Final Action 
Surplus 1974 



See 7.006-7.007, 12th ed. 



934.01 Moisture in Animal Feed 

Drying in Vacuo at 95-100° 
Final Action 

Dry amt sample contg ca 2 g dry material to const wt at 95- 
100° under pressure <100 mm Hg (ca 5 hr). For feeds with 
high molasses content, use temp. <70° and pressure ^50 mm 
Hg. Use covered Al dish >50 mm diam. and <40 mm deep. 
Report loss in wt as moisture. 

Ref.: JAOAC 17, 68(1934); 51, 467(1968); 60, 322(1977). 



925.04 Moisture in Animal Feed 

By Distillation with Toluene 
Final Action 

A. Apparatus 

Connect 250 mL flask of Pyrex or other resistant glass by 
means of Bidwell-Sterling moisture receiver to 500 mm Liebig 
condenser. Calibrate receiver, 5 mL capacity, by distg known 
amts H 2 into graduated column, and estg column of H 2 to 



69 



930.15 Moisture in Animal Feed 

Drying at 135° 
Final Action 

(Not to be used when fat detn is to be made on same sample) 

Regulate air oven to 135 ±2°. Using low, covered Al dishes, 
934.01, weigh ca 2 g sample into each dish and shake until 
contents are evenly distributed. With covers removed, place 
dishes and covers in oven as quickly as possible and dry sam- 
ples 2 hr. Place covers on dishes and transfer to desiccator to 
cool. Weigh, and calc. loss in wt as H 2 0. 

Refs.: JAOAC 13, 173(1930); 14, 152(1931); 17, 178(1934); 
18, 80(1935). 



953.07 Moisture in Animal Feed 

In Highly Acid Milk By-products 

Final Action 

Add ca 2 g ZnO, freshly ignited or oven dried, to flat-bot- 
tom dish >5 cm diam. and weigh. Add ca 1 g sample and 



70 



Animal Feed 



AOAC Official Methods of Analysis (1990) 



weigh quickly. Add ca 5 mL H 2 and distribute sample evenly 
on bottom of dish. Heat on steam bath, exposing max. surface 
of dish bottom to live steam until apparently dry. Heat at 98- 
100° in air oven 3 hr or to const wt. Cool in desiccator; then 
weigh quickly. Det. wt residue. Oil. with twice its vol. C0 2 - 
free H 2 0. Add 2 mL phthln, and titr. with 0. IN NaOH to first 
persistent pink. Calc. as % lactic acid by wt. (1 mL 0.1N 
NaOH = 0.0090 g lactic acid.). To compensate for H 2 formed 
when acid is neutzd by ZnO, add 0.1 g to residue wt for each 
g acid (as lactic) in weighed sample. Report % residue (cor- 
rected) as total solids. 

Refs.: JAOAC 36, 213(1953); 37, 253(1954). 



942.05 



Ash of Animal Feed 
Final Action 



Weigh 2 g sample into porcelain crucible and place in temp. 
controlled furnace preheated to 600°. Hold at this temp. 2 hr. 
Transfer crucible directly to desiccator, cool, and weigh im- 
mediately, reporting % ash to first decimal place. 

Refs.: JAOAC 25, 857(1942); 26, 220(1943), 



935.11* Protein in Animal Feed 

Qualitative Tests 

Final Action 
Surplus 1965 

A. Biuret Test 

See 22.012-22.013, 10th ed. 

S. Mitlon Test 
See 22.014-22.015, 10th ed. 

C. Glyoxylic Acid Test (Hopkins-Cole) 
See 22.016-22.017, 10th ed. 

D. Adamkiewicz Test 
See 22.018, 10th ed. 

E. Xanthoproteic Test 
See 22.019, 10th ed. 



954.01 Protein (Crude) in Animal Feed 

Kjeldahl Method 
Final Action 

Det. N as in 955.04. Multiply result by 6.25, or in case of 
wheat grains by 5.70. 

Refs.: JAOAC 37, 241(1954); 38, 56(1955). 



988.05 Protein (Crude) in Animal Feed 

CuS0 4 /Ti0 2 Mixed Catalyst Kjeldahl Method 
First Action 1988 

(Caution: See safety notes on sulfuric acid and sodium 
hydroxide.) 

A. Principle 

Sample is digested in H 2 S0 4 , using CuS0 4 /Ti0 2 as cata- 
lysts, converting N to NH 3 which is distd and titrd. 



B. Reagents 

(a) Sodium hydroxide soln. — Dissolve ca 450 g NaOH pel- 
lets or flakes (low N) in H 2 0, cool, and dil. to 1 L; or use 
soln with sp. gr. >1.36. 

(b) Boiling stones. — Alundum, 8-14 mesh (No. 1590-D18; 
Thomas Scientific Co.). 

(c) Methyl red indicator. — Dissolve 1 g Me red (Na salt) in 
100 mL MeOH. 

(d) Hydrochloric or sulfuric acid std soln. — 0.5N. Prep, as 
in 936.15 or 890.01. 

(e) Sodium, hydroxide std soln. — 0.1N. Prep, as in 936.16. 

After stdzg both acid and base by methods suggested in (d) 
and (e), also check one against the other. In addn, check entire 
method by analyzing NIST Std Ref. material No. 194, 
NH 4 H 2 P0 4 , certified 12.15% N, and a high purity JysineHCl. 

C. Apparatus 

(a) Digestion.— Kjeldahl flasks with capacity of 500-800 
mL. 

(b) Distillation. — Digestion flask (e.g.. Corning Glass No. 
2020) connected to distn trap by rubber stopper. Distn trap is 
connected to condenser with low-S tubing. Outlet of condenser 
tube should be <4 mm diam. 

D. Determination 

Weigh 0.250-1 .000 g sample into digestion flask. Add 16.7 
g K 2 S0 4 , 0.01 g anhyd. CuS0 4 , 0.6 g Ti0 2 , 0.3 g pumice, 
0.5-1.0 g Alundum granules, and 20 mL H 2 S0 4 . (Add addnli 
1.0 mL H 2 S0 4 for each 0.1 g fat or 0.2 g other org. matter if 
sample wt is >1 g.) 

Include at least 1 sample of high purity lysine -HO in each 
day's run as check of correctness of digestion parameters. If 
recovery is not complete, make appropriate adjustments. 

To digest sample, first adjust heat to bring 250 mL H 2 at 
25° to rolling boil in 5 min. Add a few boiling chips to prevent 
superheating. Then heat samples at this 5 -min boil rate until 
dense white fumes clear bulb of flask (ca 10 min), swirl gently, 
and continue heating addn] 40 min. (Note: Reagent propor- 
tions, heat input, and digestion time are critical factors — do 
not change.) Cool, cautiously add about 250 mL H 2 0, and 
cool to room temp. (Note: Add H 2 as soon as possible to 
reduce amt of caking. If excessive bumping occurs during distn, 
increase diln H 2 from 250 mL to ca 300 mL.) 

Prep, titrn beaker by adding appropriate vol. of acid std soln 
to amt of H 2 such that condenser tip will be sufficiently im- 
mersed to trap all NH 3 evolved. Add 3-4 drops of indicator 
soln (c). 

Add addnl 0.5- L0 g Alundum granules to cooled digestion 
flask. Optionally, 2-3 drops of tributyl citrate may also be 
added to reduce foaming. Slowly down side of flask, add suf- 
ficient NaOH soln (a) such that mixt. will be strongly alk. 
Immediately connect flask to distn app., mix completely, and 
distill at ca 7.5-min boil rate until >150 mL distillate is col- 
lected in titrn beaker. 

Titr. excess std acid in distillate with NaOH std soln (e). 
Correct for blank detn on reagents. Calc. % nitrogen: 

%N = {[(N acid )(mL acid ) - (mL bk )(N NilOH ) 

- (mL Na oH)(N Na o H )] x 1400.67}/mg sample 

where mL NaOH = mL std base needed to titr. sample; mL acid 
~ mL std acid used for that sample; mL bk - mL std base 
needed to titr. 1 mL std acid minus mL std base needed to 
titr. reagent blank carried thru method and distd into 1 mL std 
acid; N acid = normality of std acid; N Nu0 h - normality of std 
base. Calc. % crude protein, defined as 6.25 x % nitrogen, 
or 5.7 x % nitrogen for wheat grains. 

Ref.: JAOAC 70, 907(1987). 



AOAC Official Methods of Analysis (1990) 



Protein 



71 



968.06 Protein (Crude) in Animal Feed 

Dumas Method 

First Action 1968 
Final Action 1969 

A. Principle 

N, freed by pyrolysis and subsequent combustions, is swept 
by C0 2 carrier into nitrometer. C0 2 is absorbed in KOH and 
vol. residual N is measured and converted to equiv. protein 
by numerical factor. 

B. Apparatus and Reagents 

(a) Nitrogen analyzer and accessories . — Consists of com- 
bustion and collection and measuring systems. Suitable instru- 
ment, Model 29 A, with following accessories and reagents is 
available from Oak Brook Instruments Div. of Perkin Elmer 
Corp., 2000 York Rd, Oak Brook, IL 60521 (Perkin Elmer's 
current model of nitrogen analyzer is PE2410N nitrogen ana- 
lyzer): Al combustion boats, No. 29-412; Vycor combustion 
tubes, No. 29-328; CuO-Pt catalyst (CuO wire form with 2.5% 
Pt reforming catalyst), No. 29-160; reduced Cu wire, No. 29- 
120; Co,0 4 , No. 29-170; CuO powder, fines, No. 29-140; 
45% KOH, No. 29-110. 

(b) Balance. — Accurate to 0.01 mg. 

(c) Barometer. — Hg type, readable to 0.1 mm. 

C. Preparation of Samples 

Grind to pass No. 30 sieve. Store in capped bottles. 

D. Determination 

Operate instrument in accordance with instructions of man- 
ufacturer. (Following directions apply to Coleman Model 29 A 
Nitrogen Analyzer. Consult Operating Directions D-360B, 
Coleman Cat. No. 29-904, for addnl details.) 

After combustion furnaces have come to thermal equilib- 
rium, turn combustion cycle control to START and let proceed 
normally thru cycle. Observe indicated temp, on pyrometer of 
both upper and lower combustion furnaces at end of combus- 
tion portion of cycle. Furnace temps should be 850-900°. If 
not, adjust. 

Prep, combustion tube by inserting stainless steel screen in 
lower end of combustion tube (end farthest from trademark). 
In upper end, place enough glass wool to form 6 mm plug 
when packed. With 11 mm glass rod, drive glass wool down 
to stainless steel plug. Holding tube vertically, pour CuO-Pt 
catalyst directly from dispenser bottle into combustion tube until 
it reaches upper end of trademark. Tap or vibrate tube on bench 
until reagent settles to approx. center of trademark. 

Weigh and record wt of empty Al combustion boat. Place 
sample in boat. Weigh and record wt of sample and combus- 
tion boat. Difference between wts is sample wt. Use following 
sample wts (mg) as guides to suitable sample sizes: bermuda 
grass 150-300; rice bran, wheat shorts, dehydrated alfalfa 150- 
250; range feed 100-200; cottonseed meal 75-150; edible soy 
protein 50-150. Weigh sample to nearest 0.01 mg. To avoid 
wt changes, record wt within 1 min after sample and boat are 
placed on balance. If this is impossible, weigh sample inside 
weighing bottle, such as Kimble No. 15165 or 15166. 

Turn combustion tube to horizontal, and carefully insert loaded 
sample boat into open end of tube. Slide or push boat, without 
spilling contents, until it reaches trademark. Raise open end 
until tube forms 60-70° angle to horizontal. Tap or vibrate 
combustion tube on bench top while rotating tube between thumb 
and forefinger. Raise open end of tube and add vol. Co 3 4 and 
vol. CuO fines equal to vol. sample. For convenient means of 
adding above reagents to samples, place vol. CuO fines and 
vol. C03O4, each equal to vol. sample, in addnl combustion 
boat; add contents of boat, but not boat itself, to combustion 



tube; and rotate partially filled combustion tube between thumb 
and forefinger while varying angle of tube 20-45° from hor- 
izontal. Continue rotating, tapping, and vibrating until sample 
is dispelled from boat and is thoroly mixed with oxidizing agents. 
Raise open end until tube forms 60-70° angle to horizontal; 
add CuO-Pt catalyst ca 12 mm above sample boat. Tap or 
vibrate gently to eliminate voids. Add CuO-Pt catalyst to within 
20 mm of top of tube, again tapping or vibrating gently to 
eliminate voids. 

Install prepd combustion tube in N analyzer. Adjust 45% 
KOH soln meniscus to calibrating mark in nitrometer with dig- 
ital readout meter. Record counter reading, /?,. (Counter read- 
ing should preferably lie between 500 and 1000 uX at this 
point. Vent control may be used to assist in arriving at this 
counter setting, if necessary.) Record syringe temp., t 1; indi- 
cated on special scale thermometer. Add 2 min more to com- 
bustion portion of cycle by turning auxiliary timer to setting 
3. (Once this is done, addnl 2 min will be automatically pro- 
grammed into each subsequent cycle.) Turn combustion cycle 
control to START. Let analyzer proceed thru its cycle. After 
cycle is complete and combustion cycle control has entered 
STAND-BY section, readjust KOH meniscus to calibration mark 
with digital readout counter. Record new counter reading, R 2 , 
and syringe temp., t 2 . Det. blank for instrument under same 
conditions as actual analysis except omit sample. 

E. Calculations 

(a) Record observed N vol., V — R 2 ~ R\, where V = 
observed N vol. (|JiL), R } = initial counter reading, and R 2 = 
final counter reading. 

(b) Det. corrected N vol. (in |xL), V c = V - (V b + V t ), 
where V b = vol. blank (|ulL), V t = vol. correction for temp. 
(|ulL) = C f (t 2 - tO. C r is obtained from Table 968.06A (based 
on final counter reading); t 2 and tj are in °K. 

(c) Det. corrected barometric pressure, P c = P - (P b + 
P v ), where P Q — observed barometric pressure (mm Hg), P b 
= barometric temp, correction (from Table 968. 06B), and P v 
- pressure correction for vapor pressure of KOH soln (from 
Table 968.06C). 



Table 968.06A Volume Correction for Temperature Correction 
Factor (C f ) (fJtL/°K) a 



Final Counter 
Reading, 



(Nitrometers with 
Check Value) 




5000 
10000 
15000 
20000 
25000 
30000 
35000 
40000 
45000 
50000 



12 

29 

45 

62 

79 

95 

112 

129 

145 

162 

179 



1 Vol. correction, V t = C f (t 2 



Table 968.06B Barometric Temperature Correction (P b ) 





°C 




Po 


(mm 


Hg) 




Temperature, 


700-749 








750-780 


10 




1.2 








1.3 


15 




1.8 








1.9 


20 




2.3 








2.5 


25 




2.9 








3.1 


30 




3.5 








3.7 


35 




4.1 








4.3 



72 



Animal Feed 



AOAC Official Methods of Analysis (1990) 



Table 968.06C Pressure Correction (P v ) for Vapor Pressure of 
KOH (for Practical Purposes, Temp, of KOH is 
Same as Syringe) 



Temperature, °K 



P v> mm Hg 



288 
293 
298 
303 
308 
313 



4.1 
5.7 
7.4 
9.6 
12.5 
16.5 



(Note: Empirical approximation of (P h + P v ) = 11.0 will 
be satisfactorily accurate for P Q between 740 and 780 mm Hg 
and syringe temp, between 298 and 305°K.) 

(d) Calc. % N = (P c x V c x 0.0449)/(r x W), where T 
= final syringe temp, in °K and W = sample wt in mg. 

Example: 

P = 750.1 mm Hg at 25°C; W - 148.91 mg 





Start 


Finish 


Counter readings, blank 


500 uX 


524 \xL 


Counter readings, sample 


524 


6955 



t] = 302. 7°K, h = 303. 0°K, V Q - 6955 - 524 = 6431 \xL 

V, - 6431 - [24 + C f (t 2 - t,)] = 

6431 - (24 + 35 x 0.3) - 6396 |xL 

P Q - 750.1 - (3.1 x 9.6) - 737.4 

% N = (737.4 x 6396 x 0.04493)/(303.0 x 148.91) = 4.69% 
(e) Calc. % protein = % N x 6.25, or % N x 5.70 in case 
of wheat grains. 

Ret.: JAOAC 51, 766(1968). 



976.05 Protein (Crude) in Animal Feed 

Automated Kjeldahl Method 

First Action 1976 
Final Action 1977 

A. Principle 

Automation of macro Kjeldahl method is in 6 steps: sample 
and reagent addn, initial and final digestion, cooling and diln, 
NaOH addn, steam distn and titrn, and automatic pumping of 
flask contents to waste. Chemistry is carried out in macro Kjel- 
dahl flasks equipped with side arms which are rotated at 3 min 
intervals thru each successive step. 

B. Apparatus 

(a) Kjeldahl {protein/ nitrogen) analyzer. — Kjel-Foss Au- 
tomatic, Model 16210 (Foss Food Technology Corp.), orequiv. 

(b) Weighing papers. — 120 x 120 mm N-free tissues, Foss 
Food Technology Corp., or equiv. 

C. Reagents 

(a) Kjel-tabs.— Contg 5 g K 2 S0 4 and 0.25 g HgO (Foss 
Food Technology Corp.). 

(b) Kjeldahl {protein/ nitrogen) analyzer reagents.— Prep. 
following according to manufacturer's instructions: (7) Sul- 
furic acid. — 96-98%. (2) Hydrogen peroxide.— 30-35%. (3) 
Ammonium sulfate std solns. — {a) Std soln I. — Dissolve 30.000 
± 0.030 g (NH 4 ) 2 S0 4 in H 2 and dil. to 1 L with H 2 0. (b) 
Std soln //.—Dissolve 0.750 ± 0.001 g (NH 4 ) 2 S0 4 in H 2 
and dil. to 1 L with H 2 0. (4) Mixed indicator soln. — Dissolve 
1.000 g Me red and 0.250 g methylene blue in alcohol and 
dil. to 1 L with alcohol. Dil. 10 mL this soln to 1 L with H 2 0. 



(5) Sodium hydroxide -sodium thiosulfate soln. — 40% NaOH- 
8% Na 2 S 2 3 .5H 2 0. (<5) Dilute sulfuric acid soln.— 0.6%. Dil. 
30 mL 96-98% H 2 S0 4 to 5 L with H 2 0. 

D. Determination 

{Caution: See safety notes on wet oxidation, sulfuric acid, 
mercury, and peroxides.) 

Place 3 Kjel-tabs in special flask (500 mL of design com- 
patible to Foss instrument) in position 1 . Shift dispenser arm 
over flask and depress H 2 S0 4 lever, initiating simultaneous addn 
of 10 mL 30-35% H 2 2 and 12-15 mL 96-98% H 2 S0 4 (de- 
pending on fat content of sample). To flask, add accurately 
weighed sample (ca 1.0 g if <45% protein, and ca 0.5 g if 
>45% protein) wrapped in weighing paper and close lid. Flask 
automatically rotates to position 2 where sample digests 3 min, 
and then to position 3 for 3 min addnl digestion. In position 
4, flask is cooled by centrifugal blower, lid opens automati- 
cally, and 140 mL H 2 is added automatically. Flask rotates 
to position 5, where NaOH-Na 2 S 2 3 soln is automatically in- 
troduced in excess. Released NH 3 is steam distd quant, into 
200 mL tall -form titrn beaker contg 50 mL mixed indicator 
soln, and is simultaneously titrd automatically with dil. H 2 S0 4 
soln delivered by photometrically regulated syringe. Final po- 
sition of syringe is measured by potentiometer, output of which 
feeds electronic circuitry for conversion to visual display and/ 
or printout in % N or % protein with appropriate conversion 
factors. In position 6, flask is emptied. Calibrate instrument 
initially each day with aliquots of (NH 4 ) 2 S0 4 std solns and 
check periodically as stated in operating manual. 

Ref.: JAOAC 59, 141(1976). 



976.06 Protein (Crude) in Animal Feed 

Semiautomated Method 

First Action 1976 
Final Action 1977 

A. Principle 

Samples are digested in 250 mL calibrated tubes, using block 
digestor. A of NH 3 -salicylate complex is read in flowcell at 
660 nm, or NH 3 , is distd into std acid and back-titrd with std 
alkali . 

B. Apparatus 

(a) Block digestor. — Model BD-20 (Technicon Instruments 
Corp.) or Model DS-20 (Tecator, Inc., 2875C Towerview Rd, 
Herndon, VA 22071). Capable of maintaining 410° and di- 
gesting 20 samples at a time in 250 mL calibrated volumetric 
tubes constricted at top. Block must be equipped with remov- 
able shields to enclose exposed area of tubes completely at or 
above ht of constriction. 

(b) Automatic analyzer. — AutoAnalyzer with following 
modules (Technicon Instruments Corp.), or equiv.: Sampler II 
or IV with 40/hr (2: 1) cam (higher ratio cams result in carry- 
over and poorer peak sepn); proportioning pump 111; NH 3 anal, 
cartridge No. 1 16-D53I-01 (or construct equiv. manifold from 
flow diagram); AAII single channel colorimeter with 15 X 1,5- 
2.0 mm id tubular flowcell, matched 660 nm interference fil- 
ters, and voltage stabilizer; and recorder of appropriate span. 
{See Fig. 976.06.) 

C. Reagents 

(a) Phosphate -tartrate buffer soln. — pH 14.0. Dissolve 50 
g NaK tartrate and 26.8 g Na 2 HP0 4 .7H 2 in 600 mL H 2 0. 
Add 54 g NaOH and dissolve. Add 1 mL Brij-35 (Technicon 
Instruments Corp.), dil. to 1 L with H 2 0, and mix. 



AOAC Official Methods of Analysis (1990) 



Protein 



73 



ml/min 
To Sampler 1-00 H 2 Q 



10 T 
—JflStiL. 



0.32 Air 



1.20 Acid/NaCI 




40/H, 2/1 S/W 



0.16 Sample 



Modified 
AO 



10 T 



Type C Membrane 



6" DIALYZER 



ftftKL 



0.80 Acid/NaCI 



0.16 ResamPle 



0.80 Acid/NaCI 



0.42 Buffer 



0.32 Salicylate 



0.16 Hypochlorite 




120 From F/C 



COLORIMETER 

660 nm 
15 mm F/C 



FIG. 976.06 — Flow diagram for semiautomated analysis for crude protein 



(b) Sodium chloride -sulfuric acid soln. — Dissolve 200 g 
NaCl in H 2 in 2 L vol. flask. Add 15 mL H 2 S0 4 and 2 mL 
Brij-35. Oil. to vol. with H 2 and mix. 

(c) Sodium hypochlorite soln. — Dil. 6 mL com. bleach soln 
contg 5.25% available CI (Clorox, or equiv.) to 100 mL with 
H 2 and mix. Prep, fresh daily. 

(d) Sodium nitroprussi.de -sodium salicylate soln. — Dis- 
solve 150 g NaC 7 H 5 3 and 0.3 g Na 2 Fe(CN) 5 .N0.2H 2 in 600 
mL H 2 0. Add 1 mL Brij-35, dil. to 1 L with H 2 0, and mix. 

(e) Nitrogen std solns. — Prep. 6 stds by accurately weigh- 
ing (±10 mg) 59, 118, 177, 236, 295, and 354 mg (NH 4 ) 2 S0 4 
primary std (Fisher Scientific Co. No. A-938, or equiv.; dry 
2 hr at 105° before use and assume theoretical value of 21 .20% 
N after drying) into individual 250 mL digestion tubes. Pro- 
ceed as in 976.06G, beginning "Add 9 g K 2 S0 4 , 0.42 g HgO, 
and 15 mL H 2 S0 4 ..." Stds may be stored and reused until 
exhausted. 

(f) Sodium hydroxide -potassium sulfide soln. — Dissolve 400 
g NaOH in H 2 6. While still warm, dissolve 30 g K 2 S in soln, 
and dil. to 1 L. 

D. Analytical System 

If manifold is to be constructed, use clear std pump tubes 
for all air and soln flows. All fittings, coils, and glass trans- 
mission lines are A All type and size. Use glass transmission 
tubing for all connections after pump to colorimeter. Construct 
modified AO fitting on sample diln loop using AO fitting, N13 
stainless steel nipple connector, and V2" length of 0.035" id 
Tygon tubing. Insert N13 nipple approx. halfway into 0.035" 
Tygon tubing. Insert tubing into side arm of AO fitting far 
enough so resample line will not pump any air. Space pump 
tubes equally across pump rollers. Cut 0. 16 mL/min resample 
pump tube <1" at entrance before connecting to side arm of 
AO fitting. In operation, add buffer and hypochlorite solns thru 
metal side arms of A 10 type fittings; add salicylate soln, (d), 
thru metal insert to 20T coil. Air, reagents, and sample are 
combined immediately after pump thru injection fittings. 



E. Start-up 

Start automatic system and place all lines except salicylate 
line in resp. solns. After >5 min, place salicylate line in resp. 
soln and let system equilibrate. If ppt forms after addn of sal- 
icylate, pH is too low. Immediately stop proportioning pump 
and flush coils with H 2 0, using syringe. Before restarting sys- 
tem, check concns of NaCl-H 2 S0 4 soln and phosphate-tartrate 
buffer soln. 

Pump lowest concn N std soln continuously thru system >5 
min and adjust baseline control on colorimeter to read 10% 
full scale. Pump highest concn N std soln continuously thru 
system until no drift exists (usually ^10 min) and adjust "std. 
cal." control to read 85% full scale. Recorder tracings must 
be stable and show <0.3 division noise. If noisy conditions 
exist, replace dialyzer membrane. When recorder tracing in- 
dicates stable condition, immediately start sampling. 

F. Shut-Down 

Place reagent lines in H 2 0, removing salicylate line first. 
Let system wash out £20 min. 

G. Colorimetric Determination 

(Caution: See safety notes on mercury.) 

Weigh samples (See Table 976.06) into dry digestion tubes. 
Add 9 g K 2 S0 4 , 0.42 g HgO, and 15 mL H 2 S0 4 to each tube. 

Table 976.06 Sample Weight 



Protein, % 


Sample, g 


6-24 


1.5±0.1 


25-40 


1.0±0.1 


41-50 


0.8±0.1 


51-60 


0.7±0.1 


61-90 


0.5±0.01 


>90 


Weigh sample equiv. 




to 50 mg N 



74 



Animal Feed 



AOAC Official Methods of Analysis (1990) 



(Calibrated metal scoops may be used for solids.) Insert tubes 
into digestor block preheated to 410°, place shields around tubes, 
and digest 45 min. 

After digestion, remove rack of tubes from block, place in 
hood, and let cool 8-10 min, (Time depends upon air flow 
around tubes.) Direct rapid spray of H 2 6 (kitchen sink dish 
rinsing sprayer works well) to bottom of each tube to dissolve 
acid digest completely. If ppt forms, place tube in ultrasonic 
bath to aid in redissolving salt. Let cool, dil. to vol., and mix 
thoroly, Transfer portion of each sample soln to Auto Analyzer 
beaker. 

Place stds in tray in increasing order of concn, followed by 
group of samples. Analyze lowest concn std in duplicate, dis- 
carding first peak. Precede and follow each group of samples 
with std ref, curve to correct for possible drift. Analyze stds 
and samples at rate of 40/hr, 2/1 sample-to-wash ratio. Prep, 
std curve by averaging peak hts of first and second set of stds. 
Plot av. peak ht stds against N concn contained in each 250 
mL tube. 

% Protein = |(mg N/250 mL from graph) 

x 6.25 x 100]/mg sample 

H. Titrimetric Determination 

Digest as in 976. 06G. Cool 5 min and add only enough H 2 
to dissolve salts (70—75 mL). Cool and attach digestion tube 
to distn head according to manufacturer's directions. Place re- 
ceiver flask contg 25 mL std acid, 936.15A or 890.01A, and 
5-1 drops Me red indicator on platform. Condenser tip must 
be below surface of std acid soln. Add 50 mL NaOH-K 2 S soln 
to tube and steam distil vigorously until 125 mL distillate col- 
lects. Titr. excess acid with std O.LVNaOH soln, 936.16. Cor- 
rect for reagent blank. 

%N = [(mL std acid x normality acid) — (mL std NaOH 
X normality NaOH)] x 1.4007/g sample 

% crude protein ^ % N x 6.25 

Refs.: JAOAC59, 134(1976); 62, 290(1979). 



984.13 Protein (Crude) in Animal Feed 

Copper Catalyst Kjeldahl Method 
First Action 1984 

(Caution: See safety notes on sulfuric acid and sodium 
hydroxide.) 

A. Principle 

Sample is digested in M 2 S0 4 , using CuS0 4 as catalyst, con- 
verting N to NH 3 which is distd and titrd. 

B. Reagents 

(a) Sodium hydroxide, — Pellets, flakes, or soln witn sp. gr. 
>1.36, low N. Dissolve ca 450 g NaOH in H 2 0, cool, dil. to 
1 L. 

(b) Alundum. — Boiling stones, 8—14 mesh (Thomas Sci- 
entific Co., No. 1590-D18). 

(c) Methyl red indicator. — Dissolve 1 g Me red (Na salt) 
in 100 mLMeOH. 

(d) Hydrochloric or sulfuric acid std soln. — 0.5N, Prep, as 
in 936.15 or 890.01. 

(e) Sodium hydroxide std soln. — 0.1N, Prep, as in 936.16. 
After stdzg both acid and base by methods suggested in (d) 

and (e), also check one against the other. In addn, check entire 
method by analyzing N1ST Std Ref. Material No. 194, 
NH 4 H 2 P0 4 , certified 12.15% N, and high purity lysine.HCJ. 



C. Apparatus 

(a) Digestion.— Uss Kjeldahl flasks with capacity of 500- 
800 mL. 

(b) Distillation.— -Use digestion flask (Corning Glass Works, 
orequiv.) connected to distn trap by rubber stopper. Distn trap 
is connected to condenser with Jow-S tubing. Outlet of con- 
denser tube should be <4 mm diam. 

D. Determination 

Weigh 0.250-1.000 g sample into digestion flask. Add 15 
g K,S0 4 , 0.04 g anhyd, CuS0 4 , 0.5-1.0 g alundum granules, 
and 20 mL H 2 S0 4 . (Add addnl 1.0 mL H 2 S0 4 for each 0.1 g 
fat or 0.2 g other org. matter if sample wt is >1 g.) 

Include at least one sample of high purity lysine, HC1 in each 
day's run as check of correctness of digestion parameters. If 
recovery is not complete, make appropriate adjustments. 

Heat flask at 5 -min boil rate (burner preheated and adjusted 
to bring 250 mL H 2 at 25 D to rolling boil in 5 min) until dense 
white fumes clear bulb of flask, swirl gently, continue heating 
addnl 90 min. (Note: Reagent proportions, heat input, and 
digestion time are critical factors— do not change.) Cool, cau- 
tiously add 250 mL H 2 0, and cool to room temp. (Note: If 
bumping occurs daring distn, vol. of H 2 may he increased 
to ca 275 mL.) 

Prep, titrn beaker by adding accurately measured appropri- 
ate vol. std acid soln to amt of H 2 such that condenser tip 
will be sufficiently immersed. Add 3-4 drops indicator soln 
(c). 

Add 2-3 drops of tributyl citrate to digestion flask to reduce 
foaming; add another 0.5-1 ,0 g alundum granules. Slowly down 
side of flask, add sufficient NaOH soln (a) such that mixt. will 
be strongly alk. Immediately connect flask to distn app., mix 
completely, and distil at ca 7.5-min boil rate until ^.1.50 mL 
distillate is collected in titrn beaker. 

Titr. excess std acid in distillate with std NaOH soln. Cor- 
rect for blank detn on reagents. Calc. % N: 

% N = [(N add )(mL, dd ) - (mL w )(N Na0H ) 

- (mL NaOH )(N NaO M)][1400.67]/mg sample 

where mL NaOH ™ mL std base needed, to titr sample; mL acid — 
mL std acid used for that sample; mL bk = mL std base needed 
to titr. 1 mL std acid minus mL std base needed to titr. reagent 
blank carried thru method and distd into 1 mL std acid; N acid 
= normality of std acid; N basc = normality of std base. Calc. 
% crude protein, defined as 6,25 x % nitrogen, or 5.7 X % 
nitrogen for wheat grains. 

Ref.: J AOAC 67, 869(1984). 



989.03 Fiber (Acid Detergent) 

and Protein (Crude) 
in Animal Feed and Forages 

Near-Infrared Reflectance Spectroscopic Method 
First Action 1989 

(Generally applicable to detn of acid-detergent fiber and crude 
protein in any forage or feed sample) 

Method Performance: 

Crude protein: 

s R = 0.15; RSD R = 0.42% 

Ac id -detergent fiber: 

s R - 0.34; RSD R - 1.14% 

Successful use of method is based on obtaining suitable cal- 
ibration for instrument by selecting learning set of samples and 



AOAC Official Methods of Analysis (1990) 



Protein 



75 



performing calibration described in Determination (b). Four 
rules to be followed in calibration of instrument are stated in 
US DA Agriculture Handbook No. 643, p. 45 ("Near Infrared 
Reflectance Spectroscopy (NIRS): Analysis of Forage Qual- 
ity." U.S. Dep. Agric. Handb. 643, U.S. Government Print- 
ing Office, Washington, DC, 96 pp.)- In brief, rules are as 
follows: 

(7) Be certain calibration samples adequately represent pop- 
ulation to be analyzed. 

(2) Conduct accurate laboratory analyses on calibration 
samples. This step cannot be overemphasized. 

(3) Select appropriate data processing technique to extract 
pertinent information from spectra. 

(4) Select correct wavelengths. 

It may be necessary to periodically update a calibration. An- 
alyst must monitor results of method to ascertain when this is 
necessary. Test sample is usually run each day and its ana- 
lytical value is detd periodically by laboratory analysis method. 
Accuracy of any chemometric procedure is limited only by 
validity of laboratory method used to measure desired quality 
parameter for all samples and selection of appropriate learning 
set. 

A. Principle 

Random portions of prepd sample are loaded into sample 
holder of NIR spectrometer. Instrument is part of system that 
has been calibrated using representative samples from popu- 
lation to be tested. Equations selected from calibration statis- 
tics, which have been validated, are used to calc. acid-deter- 
gent fiber and crude protein content of feed and forage samples. 

B. Apparatus 

(a) Wavelength-scanning instrument, — Model 6100 or 6350 
grating monochromator (Pacific Scientific Corp., Gardner/ 
Neotec Instrument Div., Silver Spring, MD 20910), or equiv. 
Monochromator is described in detail in (I) Landa, I. Rev. 
Sci. Instrum. 50, 34-40(1979); and (2) Landa, I., & Norris, 
K.H. Appl. Spectrosc. 23, 105-107(1979). 

(b) Computer. — PDP 1 1 Series computer equipped with 64 
K bytes of main memory; dual RX02 double-density floppy 
disks; RL01 5-megabyte or RL02 10-megabyte hard disks. PDP 
system software RT-1 1 V5.0 or later (Digital Equipment Corp. , 
Nashua, NH 03061), or equiv. 

(c) USD A public software. — Software is described in detail 
in USDA Agriculture Handbook No. 643. Software consists 
of 14 programs written in FORTRAN IV to collect, store, and 
process NIRS data. Repository for software: U.S. Department 
of Agriculture, Richard B. Russell Agricultural Research Cen- 
ter, Plant Structure and Composition Research Unit, PO Box 
5677, Athens, GA 30613. Commercial software is available 
from several vendors. 

(d) Mill. — Tecator cyclone sample mill with 1 mm screen 
(Fisher Scientific Co.), or equiv. Periodically change grinding 
ring to ensure consistency of particle size over time. 

(e) NIRS sample holder. — Nylon, 2.5 cm diam., 1 cm thick, 
with IR trans mittance quartz window. Sample capacity 0.75- 
1.75 g. Sample is held in place with sep. back made of rubber 
or foam core (Pacific Scientific Corp., Gardner /Neotec In- 
strument Div.), or equiv. 

(f) Sample storage container . — For maintaining const 
moisture concn in samples. For best results, use Poly Kraft 
Bags-Mil-B-121 Type II, Grade A, Class I. Place sample in 
bag and heat-seal (EDCO Supply Corp., Brooklyn, NY 11232, 
or equiv.). 

C. instrument Operation 

(a) Start-up. — For best results, run instrument continu- 
ously. If instrument is cold, warm-up time should be >15 min 
and may require 1 h. 



(b) Monochromator diagnostic tests. — (/) Instrument 
noise. — Scan ceramic ref. to itself. Collect 25 repetitions of 
64 scans. Express deviations from zero as av. deviation (bias), 
and as root mean sq (RMS), expressed as log (1/R)/10 6 , where 
R = reflectance. Bias indicates any systematic change in log 
(1/R) level of scans taken over time. Bias values that are all 
pos. or all neg. indicate problem with instrument. RMS value 
can range from low of 10 to high of 50 without affecting anal- 
ysis. Monochromators should have av. noise level below 30 
RMS over 100 scans. 

(2) Wavelength accuracy. — Use clear polystyrene petri dish 
to measure wavelength repeatability and accuracy. Place petri 
dish in light beam and then pull out sample drawer to expose 
ceramic std. Ref. this scan to measurements without petri dish. 
Locate and compare major styrene peaks with known locations 
at 1680.3, 2164.9, and 2304.2 nm. Repeatability std dev. should 
be <0.05 nm, and accuracy from known location should 
be <0.5 nm. Large pos. values for wavelength accuracy and 
repeatability usually indicate mech. problems in monochro- 
mator. 

(c) Maintenance. — Whenever dust accumulates, use vac- 
uum, brush, or soft tissue to clean ceramic std, all parts of 
drawer assembly, and windows above and below detector. In- 
strument operation is described in detail in USDA Agriculture 
Handbook No. 643 and in Shenk, J.S., Westerhaus, M.O., & 
Hoover, M.R. Proc. Am. Soc. Agric. Eng. (1978), p. 242. 

D. Determination 

(a) Preparation of sample. — Grind samples for NIRS anal- 
ysis in cyclone mill through 1 mm screen. Clean mill between 
samples to minimize cross-contamination. Prior to grinding, 
dry samples contg >25% moisture in 60° air oven for 24 h. 
Mix milled samples well, and place random portion in sample 
holder. Continue to add random portions until NIRS sample 
holder is 2 / 3 full. Press back into holder until it is tight and 
level. As check, invert holder and make certain sample is firmly 
pressed against window. If any abnormality is apparent, re- 
move back and repeat procedure. Consistency in sample han- 
dling and prepn is crucial to successful use of NIRS technique. 

(b) Calibration. — To calibrate system for acid -detergent fi- 
ber and crude protein detns, randomly select samples that are 
representative of population to be analyzed, using either finite 
or infinite population. Finite population has defined boundaries 
set by analyst which limit population; infinite population has 
no such boundaries. Select sufficient number of samples to 
represent range of acid-detergent fiber and crude protein concns 
and all other variables that may affect chem. and physical 
composition of feed or forage (stage of growth, species, pres- 
ervation method, etc.). In practical terms, min. of 50 samples 
should be considered. 

Collect reflectance (R) measurements (log 1 /R) of calibra- 
tion samples with program SCAN, Apparatus (c), at 2.0 nm 
intervals from 1100 to 2500 nm. Develop multiterm calibra- 
tion equations by multiple linear regression of reflectance mea- 
surements to acid-detergent fiber and crude protein concns us- 
ing program CAL (c). Before regression statistics are evaluated, 
examine differences (residuals) between NIRS data and ref. 
method data for samples with large /-values. Large pos. or 
neg. /-value indicates that residual is 2.5 times std error of 
difference between NIRS detn and ref. method detn, and that 
laboratory values from ref. method were inaccurate or did not 
represent samples at time scan was taken (i.e., subsampling 
error). Reanalyze these samples by ref. method. 

In addn, evaluate output for samples that have large H-val- 
ues. Large H- statistic (>3) indicates that NIRS spectra used 
in calibration for that sample differ substantially from NIRS 
spectra of other samples. Calcn of H was mathematically de- 
rived from co variance matrix according to formula: H = 



76 



Animal Feed 



AOAC Official Methods of Analysis (1990) 



X(s 1 x)~ 1 x f [Landa. Apparatus (a)]. High value on diagonal of 
H matrix indicates sample that is dissimilar to calibration set 
at wavelengths used in equation. Rescan such samples. If 2 
scans agree and sample belongs in population, then retain sam- 
ple. If scans disagree, then first scan was mistake and should 
be discarded. 

Examine std error of calibration (SEC) to det. fit of cali- 
bration samples: the lower the SEC, the better the fit. Select 
equation with SEC about 2 times laboratory repeatability std 
dev. for acid-detergent fiber and crude protein ref. method. 
Examine coeff. of detn (R 2 ) to det. proportion of variation in 
ref. method values among samples explained by NIRS regres- 
sion equation. Low R 2 values often indicate that laboratory 
data from ref. method are imprecise. If laboratory repeatability 
error from ref. method is l U of std dev., select equation with 
R 2 >:(X75> 

Examine F-statistic of regression coeffs. High F-values in- 
dicate that regression coeff. is significantly different from zero; 
small ^-values indicate that coeff. contributes little to equation 
except to fit random errors. Probability that observed F-value 
was obtained solely through chance does not follow std F ta- 
bles because F is selected as max. of all wavelength combi- 
nations considered. As number of choices increases, large F- 
values are needed to signify coeff. fitting more than just ran- 
dom errors. Reject equations with F- values <10. 

(c) Validation. — Conduct NIRS analysis (program PRE) with 
equations selected from calibration statistics on population of 
unknown samples. Examine NIRS data for samples with larger 
H- values. Large H- value (>3.0) for a few samples indicates 
that their NIR spectra are different from spectra of calibration 
population. Large lvalue (2.5 times SED) for a few samples 
indicates that laboratory values from ref. method were inac- 
curate or did not represent samples at time scan was taken. If 
many validation samples have large t- and H-values, over- fit- 
ting has occurred, and equation is specific to samples in cal- 
ibration set. Next, use validation statistics from program STAT 
to examine std error of analysis (SEA) by NIRS of chem, com- 
position of validation samples. SEA is true indication of per- 
formance of equation on unknown samples. Select equation 
with lowest bias and SEA. Unlike SEC, which must decrease 
with each addnl term, SEA only decreases with addnl terms 
until over- fitting becomes important and forces it to increase. 
Best equation for routine NIRS analysis is based on both su- 
perior calibration and validation statistics. 

Ref.: JAOAC71, 1162(1988). 



941.04 Urea and Ammoniacal Nitrogen 

in Animal Feed 
Urease Method 
Final Action 
A. Reagents 

(a) Defoaming soln. — Dow Corning Corp. Antifoam B 
Emulsion. 

(b) Urease soln. — Prep, fresh soln by dissolving stdzd urease 
in H 2 so that each 10 mL neutzd soln will convert N of >0. 1 
g pure urea. 

Standardization, — To det. alky of com. urease prepn dis- 
solve 0.1 g in 50 mL H 2 and titr. with O.LV HC1, using Me 
red, 984.13B(c). Add same vol. O.LV HC1 to each 0.1 g urease 
in prepg urease soln. To det. enzyme activity, prep, ca 50 mL 
neutzd 1% soln. Add different amts of soln to 0.1 g samples 
pure urea and follow with enzyme digestion and distn as in 
detn. Calc. activity of urease prepn from amt of this urease 
soln that completely converted urea, as detd by complete re- 
covery of N by distn. 



(c) Calcium chloride soln. — Dissolve 25 g CaCl 2 in 100 mL 
H 2 0. 

B. Determination 

Place 2 g sample in Kjeldahl flask with ca 250 mL H 2 0. 
Add 10 mL urease soln, stopper tightly, and let stand 1 hr at 
room temp, or 20 min at 40°. Cool to room temp, if necessary. 
Use addnl urease soln if feed contains >5% urea (ca 12% pro- 
tein equiv.). Rinse stopper and neck with few mL H 2 0. Add 
>2 g MgO (heavy type), 5 mL CaCl 2 soln, and 3 mL de- 
foaming soln, and connect flask with condenser by Kjeldahl 
connecting bulb. Distil 100 mL into measured vol. std acid, 
936.15 or 890.01, and titr. with std alkali, 936.16, using Me 
red, 984.13B(c). 

Refs.: JAOAC 24, 867(1941); 25, 874(1942); 27, 494(1944). 



967.07 Urea in Animal Feed 

Coiorimetric Method 

First Action 1967 
Final Action 1970 

(Applicable to animal feeds and their ingredients) 

A. Apparatus 

Spectrophotometer . — Instrument with max. band width 2.4 
nm at 420 nm, with 1 cm cells. 

B. Reagents 

(a) p-Dimethylaminobenzaldehyde (DMAB) soln. — Dis- 
solve 16.00 g (Eastman Kodak Co. No. 95 only) in 1 L alcohol 
and add 100 mL HC1. Stable 1 month. Prep, new std curve 
with each new batch of reagent. 

(b) Zinc acetate soln. — Dissolve 22. g Zn(OAc) 2 .2H 2 in 
H 2 0, add 3 mL HOAc, and dil. to 100 mL. 

(c) Potassium ferrocyanide soln. — Dissolve 10.6 g 
K 4 Fe(CN) 6 .3H 2 in H 2 and dil. to 100 mL. 

(d) Vegetable charcoal. — Darco G-60. 

(e) Phosphate buffer soln. — pH 7.0. Dissolve 3.403 g an- 
hyd. KH 2 P0 4 and 4.355 g anhyd. K 2 HP0 4 sep. in ca 100 mL 
portions freshly distd FLO. Combine solns and diL to 1 L with 
H 2 0. 

(f) Urea std solns. — (J) Stock soln. — 5 mg/mL. Dissolve 
5.000 ± 0.001 g reagent grade urea in H 2 and dil. to 1 L 
with H 2 0. (2) Working solns.— -0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 
1.4, 1.6, 1.8, and 2.0 mg urea/5 mL. Pipet 2, 4, 6, 8, 10, 
12, 14, 16, 18, and 20 mL stock soln into 250 mL vol. flasks 
and dil. to vol. with phosphate buffer. (3) Reference soln. — 
Use std soln contg 1.0 mg urea/5 mL as ref. std. Store at 
<24°. Stable 1 week. 

C. Preparation of Standard Curve 

Pipet 5 mL aliquots of working std solns into 20 x 150 mm 
(25 mL) test tubes and add 5 mL DMAB soln to each. Prep, 
reagent blank of 5 mL buffer soln and 5 mL DMAB soln. 
Shake tubes thoroly and let stand 10 min in H 2 bath at 25°. 
Read A in 1 cm cell at 420 nm with reagent blank at zero A. 
Plot A against concn urea. Plot should be straight line; if not, 
repeat, using new lot of DMAB . 

D. Determination 

Weigh LOO g ground sample into 500 mL vol. flask. Add 
1 g charcoal, ca 250 mL H 2 0, 5 mL Zn(OAc) 2 soln, and 5 
mL K 4 Fe(CN) 6 soln. Shake mech. 30 min and dil. to vol. with 
H 2 0. Let stand until ppt settles. Decant thru Whatman No. 40 
paper and collect clear filtrate. Pipet 5 mL filtrate into test 
tube, add 5 mL DMAB soln, and shake thoroly. Include ref- 
erence std (5 mL soln (f)(3) and 5 mL DMAB soln) and re- 



AOAC Official Methods of Analysis (1990) 



Nitrogen 



77 



agent blank with each group of samples. Let stand 10 min in 
H 2 bath at 25°. Read A at 420 nm against reagent blank. 

% Urea = (1.0 x A samp]c X I00)/O4 std X mg sample in aliquot) 

Ret.: JAOAC 50, 56(1967). 

CAS-57-13-6 (urea) 



920.37* Nitrogen (Albuminoid) 

in Animal Feed 

Final Action 
Surplus 1965 



See 22.020-22.021, 10th ed. 



920.38* Nitrogen (Amido) 

in Animal Feed 

Final Action 
Surplus 1965 

% Amido N = % Total N - % albuminoid N 



968.07 Nitrogen (Nitrate and Nitrite) 

in Animal Feed 
Colorimetric Method 

First Action 1968 
Final Action 1970 

A. Principle 

Nitrate and nitrite are extd with Cd and Ba chloride soln. 
Bulk of sol. proteins are pptd in aJk. soln and clarified soln is 
passed thru metallic Cd column, reducing nitrate to nitrite. Ni- 
trite is measured colorimetrically. 

(Caution: Cd salts are toxic.) 

B. Reagents and Apparatus 

(a) Nitrate nitrogen std solns. — (I) Stock soln. — 12 |xg ni- 
trate N/mL. Dissolve 0.867 g KN0 3 in 1 L H 2 0. Dil. 25 mL 
to 250 mL with H 2 0. (2) Working solns. —0.6, 1.2, 1.8, 2.4, 
3.0 Ltg N/mL. Dil. 5, 10, 15, 20, and 25 mL stock soln to 
100 mL with H 2 0. 

(b) Extracting soln. — Dissolve and dil. 50 g CdCl 2 and 50 
g BaCl 2 to 1 L with H 2 0. Adjust to pH 1 with HC1. 

(c) Ammonium chloride buffer soln. — pH 9.6. Dissolve 50 
g NH 4 C1 in 500 mL H 2 and adjust pH with NH 4 OH. Dil. to 
1 L with H 2 0. 

(d) Sodium hydroxide soln. — 2.5/V. Dissolve 50 g NaOH 
in 500 mL H 2 CX 

(e) Sulfanilamide soln. — 0.5%. Dissolve 1.25 g sulfanila- 
mide in 250 mL HC1 (1 + 1). Soln is stable 1-2 months. 

(f) Coupling reagent. — Dissolve 0.5 g 7V-(l-naph- 
thyl)ethylenedi amine. HO in 100 mL H 2 0. Store in g-s dark 
bottle in refrigerator. Soln is stable several weeks. 

(g) Salt soln.— Dissolve 100 g NaCl in 500 mL H 2 0. Add 
50 mL buffer soJn, (c), and dil. to 1 L with H 2 0. 

(h) Reduction tube. — 25 mL buret or equiv. id chromatgc 
tube with stopcock and reservoir (Kontes Glass Co. Cat. No. 
K-420280 or Lurex Scientific Cat. No. JC-1506). 

C. Preparation of Columns 

Prep, supply of metallic Cd by placing Zn rods into 500 mL 
20% CdS0 4 soln. After reaction for 3 hr, discard soln and 
scrape moss- 1 ike Cd growth from Zn rods. Place Cd in high- 



speed blender, add 500 mL H 2 0, and blend 2 sec. Wash fine 
metal particles with H 2 onto sieves, collecting only 20™40 
mesh size. Fill reduction tube with H 2 and add 2 cm plug of 
glass wool. Press any trapped air from glass wool as it is pushed 
to bottom of column with glass rod. Add Cd to depth of 10 
cm, using min. of very gentle tapping. Wash column with 25 
mL 0.107V HC1, two 25 mL portions H 2 0, and finally 25 mL 
buffer, (c), dild 1 + 9. Keep column covered with salt soln, 
(g), when not in use. 

Normally columns can be used repeatedly if kept under salt 
soln between analyses. When succession of highly proteina- 
ceous or other sol. org. contg samples are treated, flow rate 
may decrease gradually. Repeating 25 mL 0.1 0A^ HC1 treat- 
ment may restore original flow rate; if not, prep, new column. 
Reproducible flow rate is important. Actual rate can be 3-5 
mL/min but once established, it must be identical (±0.1 mL) 
for samples and stds. 

D. Preparation of Standard Curve 

Prep, std curve of 3, 6, 9, 12, and 15 \xg nitrate- nitrite N 
by pipeting 5.0 mL aliquots of working std solns into 30 mL 
beakers. Add 5 mL buffer soln, (c), and 1.5 mL H 2 0, mix 
well, and transfer quant, to reduction column, using min. H 2 0. 
Adjust flow rate thru column to 3-5 mL/min. Just as reservoir 
empties, add 15 mL salt soln, (g). Collect eluate, including 
salt wash, in 50 mL vol. flask (total vol. of eluate should be 
ca 40 mL). Add 5 mL sulfanilamide soln, (e), mix, and let 
stand 3 min. Add 2 mL coupling reagent, (f), mix, dil. to vol. 
with H 2 0, mix, and let stand 20 min for max. color devel- 
opment. Color is stable ^2 hr. Det. A in 1 cm cells at 540 
nm against reagent blank. Plot A against |xg nitrate-nitrite N. 

E. Extraction 

(a) Low level nitrate samples (grains, meals, supplements, 
etc.). — Wash 5.0 g finely ground sample into 250 mL vol. 
flask. Add 100 mL extg soln, (b), and 100 mL H 2 0, and mix. 
Let stand 1 hr with occasional swirling. Add 20 mL 2.5A NaOH, 
dil. to vol. with H 2 0, mix, and filter immediately thru rapid 
paper. Pipet 10 mL buffer soln, (c), into 100 mL vol. flask, 
dil. to vol. with clear filtrate, and mix. 

(b) Dry, high level nitrate products (dried plants, hays, 
meals, etc.). — Weigh 5.0 g finely ground sample into 500 mL 
vol. flask. Add 100 mL extg soln, (b), and 300 mL H 2 0, and 
mix. Let stand 1 hr with occasional swirling, add 40 mL 2.5N 
NaOH, dil. to vol. with H 2 0, mix, and filter immediately thru 
rapid paper. Pipet 10 mL buffer soln, (c), into 100 mL vol. 
flask, dil. to vol. with clear filtrate, and mix. 

(c) Grasses, silages, and other wet materials. — Weigh 100 
g sample into I gal. capacity high-speed blender. Add 100 mL 
extg soln, (b), and 800 mL H 2 0, including vol. H 2 contrib- 
uted by sample as detd in 934.01 or 925. 04B. Homogenize 1 
min, pour into 2 L beaker, and let stand 1 hr. Add 100 mL 
buffer soln, (c) (total vol. 1 L), mix well, and filter thru What- 
man No. 42 paper, collecting portion of clear filtrate. 

F. Determination 

(a) Nitrate plus nitrite nitrogen. — Pipet 25 mL buffered 
sample exts, 968.07E(a) or (b), or 5 mL ext, (c), into reduc- 
tion column and treat as in 968. 07D, beginning, "Adjust flow 
rate thru column ..." Rinse column with 30 mL H 2 be- 
tween samples to remove NaCI. Use portion of buffered sam- 
ple exts with equiv. diln and pH as ref. soln in detg A at 540 
nm. Also det. nitrate- nitrite in reagents and correct for this 
blank value. Calc. total nitrate- nitrite N from std curve. 

(b) Nitrite nitrogen. — Pipet aliquot clear sample filtrate 
(contg <15 fxg nitrite) into 50 mL vol. flask and dil. with H 2 
to ca 40 mL. Mix well, add 5 mL sulfanilamide soln, (e), mix, 
and let stand 3 min. Add 2 mL coupling reagent, (f), and dil. 



78 



Animal Feed 



AOAC Official Methods of Analysis (1990) 



to vol. with H 2 0. Mix we J J and let stand 20 min for max. color 
development. Measure A in 1 cm cells against sample ext with 
equiv. diln at 540 nm. Correct for nitrite reagent blank. 

(c) Nitrate nitrogen. — Calc. by difference between (a) and 
(b) above. 

G. Calculation 

ppra NO r N and/or NO3-N 

— fxg N0 3 -N found X diln factor/g sample 

Diln factors for exts: 968.07E(a), 11.1; (b), 22.2; (c), 200. 

Ref.: JAOAC 51, 763(1968). 



971.09 Pepsin Digestibility 

of Animal Protein Feeds 
Filtration Method 

First Action 1971 
Final Action 1973 

A. Principle 

Defatted sample is digested 16 hr with warm soln of pepsin 
under const agitation. Insol. residue is isolated by filtering, 
washed, dried, and weighed to det. % residue. Residue is ex- 
amined microscopically and analyzed for protein. Filtration 
method is applicable to all animal proteins. Methods are not 
applicable to vegetable proteins or mixed feeds because of 
presence of complex carbohydrates and other compds not di- 
gested by pepsin. 

B. Apparatus 

(a) Agitator. — See Fig. 971.09. Continuous, slow speed (1.5 
rpm), end-over-end type, to operate inside incubator at 45 ± 
2° and carry 8 oz screw-cap prescription bottles, or equiv. Ag- 
itator and bottles available from D. E. Sims, 716 Forrest Ave, 
Quincy, IL 62301. Stirring or reciprocating (shaking) type ag- 
itator cannot be used because solid particles collect on sides 
of bottle and do not contact pepsin soln. If heat from agitator 
motor raises incubator temp, to >45°, mount motor outside 
incubator by drilling hole thru side of incubator and connecting 
motor to agitator with extension shaft and coupling (available 
from agitator supplier). (Caution: See safety notes on electri- 
cal equipment.) 

(b) Settling rack. — Wood or metal to hold digestion bottles 
at 45° angle. May be made from 2 boards nailed horizontally 




FIG. 971. 09— Agitator 



into "V" cut into vertical end pieces. Also available from ag- 
itator supplier, (a). 

(c) Filtering device. — Modified California buchner, 
962.09C(d), available from Labconco Corp., 8811 Prospect 
Ave, Kansas City, MO 64132, No. 55100. (If edge of screen 
is rough, smooth with small -tip soldering iron.) Use with re- 
tainer sleeve, 2 X 2.75" od stainless steel tube, available from 
agitator supplier, (a). 

(d) Glass fiber filter. — 7 cm, Whatman, Inc., No. 934-AH, 
or equiv. 

(e) Moisture dishes. — Al, 78 mm od X 20 mm, with out- 
side cover and vertical sides (Curtin Matheson Scientific, Inc., 
No. 19370-30, or equiv.). 

C. Reagent 

Pepsin soln. — 0.2% pepsin (activity 1:10,000) in 0.075/V 
HC1; do not use pepsin NF or pepsin of activity other than 
1 : 10,000. Prep, just before use by dilg 6.1 m'L HCI to 1 L 
and heating to 42-45°. Add pepsin and stir gently until dis- 
solved. Do not heat pepsin soln on hot plate or overheat. 

D. Preparation of Sample 

Sieve sample, 965.16, thru No. 20 sieve. Grind portion re- 
tained on sieve to pass No. 20 sieve. Combine both portions 
and blend by stirring and shaking in pt (500 mL) jar. Thoro 
blending is essential. Because of high fat content of many an- 
imal products, grinding without sieving may cause sticking in 
mill, loss of moisture or fat, or poorly blended sample. 

E. Extraction 

{Caution: See safety notes on distillation, flammable solvents, 
and diethyl ether.) 

Prep, extn thimble from 1 1 cm Whatman No. 2 paper, or 
equiv., as follows: Fold paper in half; straighten paper and 
refold at right angles to first fold; turn paper over and repeat 
process with folds at 45° to original fold; while holding creased 
paper in one hand, place short test tube (6-8 mm smaller in 
diam. than extractor sample holder or cup in which thimble is 
to be used) at its center; fold along natural crease lines to form 
4-pointed star around tube; and wrap points in same direction 
around tube to complete thimble. 

Weigh 1.000 g ground sample (0.500 g of poultry byprod- 
ucts or hydrolyzed feathers because of gummy nature and amt 
of residue) into thimble and ext I hr with ether at condensation 
rate of 3-4 drops /sec. (If Soxhlet is used, top of thimble should 
extend above siphon tube to avoid loss of solid particles. If 
paper contg sample is totally submerged in siphon cup, sample 
must be completely wrapped in paper.) Observe ether ext to 
det. that no solid particles were carried into sol v. For approx. 
fat content detn, evap. ether, and dry and weigh residue. Re- 
move paper from sample container or cup and let dry at room 
temp. Unfold, and quant, brush defatted sample into digestion 
bottle, avoiding contamination by brush bristles or filter paper 
fibers. Use of powder funnel is helpful to avoid loss. 

F. Pepsin Digestion 

To defatted sample in agitator bottle add 150 mL freshly 
prepd pepsin soln pre warmed to 42-45°. Be sure sample is 
completely wetted by pepsin soln. Stopper bottle, clamp in 
agitator, and incubate with const agitation 16 hr at 45°. 

G. Treatment of Residue 

Dry individual sheets of glass fiber filter, (d), 30 min at 1 10° 
in moisture dishes with cover open. Cool in desiccator 30 min 
with cover closed, and weigh (Wj). 

Remove bottles from agitator. Place in 45° angle settling 
rack and loosen caps. Let residue settle > 15 min. Place weighed 
filter in California buchner, (c), apply suction, and moisten 



AOAC Official Methods of Analysis (1990) 



Fat 



79 



with fLO. Place retainer sleeve on filter and press down gently. 
Rinse particles of residue on cap onto filter with small amt 
H 2 0. Carry bottle from rack to filter at same angle as settled 
and slowly pour contents thru filter as continuous small stream, 
avoiding all unnecessary agitation. Liq. passes thru paper as 
rapidly as poured, with residue spreading over surface of filter 
but not covering it completely until all or practically all of liq. 
has passed thru. If filtration rate becomes slow, it may be ac- 
celerated by adding acetone washes described below, but only 
if no significant amt of digestion mixt. remains on funnel when 
acetone is added. (Filtration (passage of aq. mixt. thru filter) 
should be complete within 1 min with most proteins.) After 
supernate has passed thru filter, quant, transfer residue onto 
filter as follows: 

Add 15 mL acetone to bottle. Hold thumb over bottle neck 
and shake vigorously. Release pressure, replace thumb over 
bottle neck, and shake bottle in inverted position over filter. 
Remove thumb, letting acetone and residue discharge onto fil- 
ter. Repeat rinse with second 15 mL portion acetone, shaking 
and releasing pressure as above. Inspect bottle, and rinse fur- 
ther with acetone, using policeman, if necessary. If >3 mm 
liq. remains on paper when acetone washes are started, it may 
be necessary to use three 15 mL acetone washes instead of 2 
to increase filtration rate. 

After all liq. passes thru funnel, wash residue and inside 
surface of retainer sleeve with 2 small portions acetone from 
wash bottle or hypodermic syringe, and suck dry. Remove re- 
tainer sleeve from funnel. Transfer filter to original moisture 
dish. Scrape or brush any residue particles or filter clinging to 
retainer sleeve or funnel onto filter in moisture dish. Dry in 
oven, cool, and weigh as before (W 2 ). Calc. % indigestible 
residue = (W 2 ~~ W,) x 100/g sample. 

Det. indigestible protein by transferring filter contg residue 
directly to Kjeldahl flask. Proceed as in 954.01. (Caution: Vi- 
olent reaction may take place when NaOH is mixed with dild 
digestion mixt., caused by large excess H 2 S0 4 due to small 
amt org. material from residue and none from glass filter. Avoid 
by thoroly mixing and cooling digestion mixt. before addn of 
NaOH or by using 20 mL H 2 S0 4 in Kjeldahl digestion instead 
of 25 mL.) Make blank detn on 1 sheet of glass filter and 
subtract from each sample detn, if necessary. Calc. % protein 
based on original sample wt. Result represents % indigestible 
protein in sample. Convert to % crude protein content of sam- 
ple not digested, "protein indigestible" = % indigestible pro- 
tein in sample x 100/% total crude protein in sample. 

Refs.: J. Agric. Food Chem. 3, 159(1955). JAOAC 40, 
606(1957); 41, 233(1958); 42, 231(1959); 43, 320 
(1960); 54, 669(1971); 55, 702(1972). 



920.39 Fat (Crude) or Ether Extract 

in Animal Feed 
Final Action 



Use method 920.39A or 920.39C for mixed feeds other than 
(/) baked and/or expanded, (2) dried milk products, or (3) 
contg urea. 

A. Indirect Method 

Det. moisture as in 934.01 or 920.36*; then ext dried sub- 
stance as in 920. 39C, and dry again. Report loss in wt as ether 
ext. 

Direct Method 

B. Reagent 

Anhydrous ether. — Wash com. ether with 2 or 3 portions 
fLO, add solid NaOH or KOH, and let stand until most of 



H 2 is abstracted from the ether. Decant into dry bottle, add 
small pieces of carefully cleaned metallic Na, and let stand 
until H evolution ceases. Keep ether, thus dehydrated, over 
metallic Na in loosely stoppered bottles. (Caution: See safety 
notes on sodium metal and diethyl ether.) 

C. Determination 

(Large amts H 2 OsoL components such as carbohydrates, urea, 
lactic acid, glycerol, and others may interfere with extn of fat; 
if present, ext 2 g sample on small paper in funnel with five 
20 mL portions H 2 prior to drying for ether extn. Caution: 
See safety notes on monitoring equipment, distillation, and 
diethyl ether.) 

Ext ca 2 g sample, dried as in 934.01 or 920.36*, with an- 
hyd. ether. Use thimble with porosity permitting rapid passage 
of ether. Extn period may vary from 4 hr at condensation rate 
of 5-6 drops /sec to 16 hr at 2-3 drops /sec. Dry ext 30 min 
at 100°, cool, and weigh. 

Refs.: JAOAC 64, 351(1981); 65, 289(1982). 



954.02 Fat (Crude) or Ether Extract 

in Pet Food 
Gravimetric Method 
Final Action 1977 

(To be used only on products which have been baked and /or 
expanded, and on intermediate moisture pet foods. Not appli- 
cable to canned, fresh, or frozen pet food. Such products should 
be dried at 70-1 10°, then ground, and drying completed by 
934.01 or 920.36* followed by 920.39A or 920.39C.) 

(Caution: See safety notes on distillation, diethyl ether, and 
petroleum ether.) 

Place ca 2 g, accurately weighed, ground, well mixed sam- 
ple in Mojonnier fat-extn tube, add 2 mL alcohol to prevent 
lumping on addn of acid, and shake to moisten all particles. 
Add 10 mL HC1 (25+11), mix well, and set tube 30-40 min 
in H 2 bath at 70-80°, shaking frequently. Cool to room temp, 
and add alcohol until liq. level rises into constricted portion 
of Mojonnier tube. 

Add 25 mL ether, stopper with glass, Neoprene, or good 
quality rubber stopper thoroly cleaned with alcohol, and shake 
vigorously 1 min. Carefully release pressure so that no sol v. 
is lost. Wash adhering sol v. and fat from stopper back into 
extn tube with few mL redistd pet ether (bp <60°). Add 25 
mLredistd pet ether, stopper, and shake vigorously 1 min. Let 
stand until upper liq. is practically clear or centrf. 20 min at 
ca 600 rpm. Pour as much of ether-fat soln as possible thru 
filter consisting of cotton pledget packed just firmly enough 
in funnel stem to let ether pass freely into 1 50 mL beaker contg 
several glass beads. Rinse lip of tube with few mL pet ether. 
Re-ext liq. remaining in tube twice, each time with only 15 
mL of each ether, shaking 1 min after addn of each ether. Pour 
clear ether soln thru filter into same beaker as before, and wash 
tip of tube, stopper, funnel, and end of funnel stem with few 
mL of mixt. of 2 ethers (1 + 1). Evap. slowly on steam bath 
under gentle stream of air or N. Continue heating on steam 
bath 15 min after solv. has evapd; then cool to room temp. 

Redissolve dried fat residue in four 10 mL portions Et ether, 
filtering each portion thru small pledget of cotton into 100 mL 
beaker, contg few glass beads, that has been predried 30 min 
at 100°, cooled to room temp, in desiccator, and weighed im- 
mediately. Use fifth 10 mL portion ether for rinsing cotton and 
funnel. Evap. ether on steam bath, dry 90 min at 100°, cool 



80 



Animal Feed 



AOAC Official Methods of Analysis (1990) 



to room temp, in desiccator, and weigh immediately. Correct 
this wt by blank detn on reagents used. 

Refs.: JAOAC 37, 250(1954); 38, 225(1955); 59, 1218(1976); 
60, 322(1977); 65, 456(1982). 



932.02 Fat (Crude) or Ether Extract 

in Dried Milk Products 
Final Action 

Proceed as in 932.06A(b) and 932.06B, using 8.5 mL H 2 
and 1.5 mLNH 4 OH. 

Refs.; JAOAC 15, 524(1932); 17, 190(1934); 18, 351(1935); 
28, 80(1945). 



948.04 Fat (Crude) or Acetone Extract 

in Fish Meal 

See 948.16 and 969.24. 



962.09 Fiber (Crude) in Animal Feed 

Ceramic Fiber Filter Method 

Rrst Action 1962 

Final Action 1971 

Revised First Action 1982 

AOCS-AOAC Method 

A. Principle 

Crude fiber is loss on ignition of dried residue remaining 
after digestion of sample with 1.25% H 2 S0 4 and 1.25% NaOH 
solns under specific conditions. Method is applicable to grains, 
meals, flours, feeds, and fiber-bearing material from which fat 
can be extd to leave workable residue. 

B. Reagents 

(a) Sulfuric acid soln,— 0.255 ± 0.005^. 1.25 g H 2 S0 4 / 
100 mL. Concn must be checked by titrn. 

(b) Sodium hydroxide soln.— 0,313 ± 0.0057V. 1.25 g 
NaOH/ 100 mL, free, or nearly so, from Na 2 C0 3 . Concn must 
be checked by titrn. 

(c) Prepared ceramic fiber. — Place 60 g ceramic fiber 
(Cerafiber, 8 Ib/cu ft, E. J. Bartell Co., 700 Powell Ave, S.W., 
Renton, WA 98055) in blender, add 800 mL H 2 0, and blend 
1 min at low speed. 

Det. blank by treating ca 2 g (dry wt) of prepd ceramic fiber 
with acid and alkali as in detn. Correct crude fiber results for 
any blank, which should be negligible (ca 2 mg). 

(d) Alcohol.- — 95% or reagent alcohol, MeOH, or isopro- 
panol. 

(e) Antifoam. — Dow Corning Corp. Antifoam A compd dild 
1+4 with mineral spirits or pet ether, or H 2 0-dild Antifoam 
B Emulsion (1+4). Do not use Antifoam Spray. 

(f) Bumping chips or granules.— -Broken Alundum cruci- 
bles or equiv. granules (RR Alundum 90 mesh, Norton Co., 
1 New Bond St, Worcester, MA 01606) are satisfactory. 

C. Apparatus 

(a) Digestion apparatus, — With condenser to fit 600 mL 
beaker, and hot plate adjustable to temp, that will bring 200 
mL H 2 at 25° to rolling boil in 15±2 min. (Available from 
Labconco Corp., 881 1 Prospect Ave, Kansas City, MO 64132.) 

(b) Ashing dishes. — Silica, Vitreosil 70 X 16 mm; or por- 
celain, Coors Ceramics Co., 600 9th St, Golden, CO 80401, 
No. 60230, or equiv. 



(c) Desiccator. — With efficient desiccant such as 4-8 mesh 
Drierite (CaCl 2 is not satisfactory). 

(d) Filtering device. — With No. 200 type 304 or 316 stain- 
less steel screen (C-E Tyler, Inc., 3200 Bessemer City Rd, 
Hwy 274, PO Box 8900, Gastonia, NC 28053), easily washed 
free of digested residue. Either Oklahoma State filter screen 
(see Fig. 962.09A; available from Labconco Corp.) or mod- 
ified California plastic buchner. (See Fig. 962. 09B; consists 
of 2 piece polypropylene plastic funnel manufactured by Nalge 
Co., 75 Panorama Creek Dr, PO Box 20365, Rochester, NY 
14602, Cat. No. 4280-0700, 70 mm (without No. 200 screen), 
or equiv. (also available from Labconco Corp.). Seal screen 
to filtering surface of funnel, using small-tip soldering iron.) 

(e) Suction filter. — To accommodate filtering devices. At- 
tach suction flask to trap in line with aspirator or other source 
of vac. with valve to break vac. 

(f) Liquid preheater . — For preheating H 2 0, 1 .25% H 2 S0 4 , 
and 1.25% NaOH solns to bp of H 2 0. Convenient system, 
shown in Fig. 962. 09C, consists of sheet Cu tank with 3 coils 
of 3 /s" (10 mm) od Cu tubing, 12.5' (3.8 m) long. Solder inlets 
and outlets where tubing passes thru tank walls. Connect to 
reflux condenser and fill with H 2 0. Keep H 2 boiling with 
two 750 watt thermostatically controlled hot plates. Use Tygon 
for inlet leads to reservoirs of H 2 0, acid, and alkali; use gum 
rubber tubing for outlets. Capacity of preheater is adequate for 
60 analyses in 8 hr. 

D. Preparation of Sample 

Reduce sample (riffle is suitable) to 100 g and place portion 
in sealed container for H 2 detn. Immediately det. H 2 0. Grind 
remainder to uniform fineness. (Weber mill (Sargent-Welch 
Scientific Co. S-60870) with screen 0.033-0.040" (No. 18 or 
20), Micro mill (Mikropul, Div. of Hosokawa Micron Inter- 
national, Inc., 10 Chatham Rd, Summit, NJ 07901) with screen 
V25-V16" (No. 18-No. 12), and Wiley mill with 1 mm (No. 
18) screen give comparable fineness.) Since most materials 
lose moisture during grinding, det. H 2 on ground sample at 
same time sample is taken for crude fiber detn. 

E Determination 

Ext 2 g ground material with ether or pet ether (initial boil- 
ing temp., 35-38°; dry-flask end point, 52-60°; >95% distg 




200 mesh st. steel screei 

20 go, st. steel back plate' 
with 5/64 1 ' holes 




FIG. 962.09A— Oklahoma State filter screen 



AOAC Official Methods of Analysis (1990) 



Fiber 



81 




FIG. 962.09B— Modified California State buchner funnel, 2-piece 

polypropylene plastic, covered with 200-mesh screen, A, 

heat-sealed to edge of filtering surface 

<54°, and <60% distg <40°; sp gr at 60°F, 0.630-0.660; evapn 
residue <0.002% by wt). If fat is <1%, extn may be omitted. 
Transfer to 600 mL beaker, avoiding fiber contamination from 
paper or brush. Add ca 1.5-2.0 g dry wt of prepd ceramic 
fiber, 200 mL boiling 1 .25% H 2 S0 4 , and 1 drop dild antifoam. 
(Excess antifoam may give high results; use only if necessary 
to control foaming.) Bumping chips or granules may also be 
added. Place beaker on digestion app. with preadjusted hot 
plate and boil exactly 30 min, rotating beaker periodically to 
keep solids from adhering to sides. Remove beaker, and filter 
as in (a) or (b). 

(a) Using Oklahoma filter screen . —Turn on suction and in- 
sert screen (precoated with ceramic fiber if extremely tine ma- 
terials are analyzed) into beaker, keeping face of screen just 
under surface of liq. until all liq. is removed. Without breaking 
suction or raising filter, add 50-75 mL boiling H 2 0. After 




FIG. 962.09C— Continuous heater for distilled water, 1.25% 
alkali, and 1.25% acid 



wash is removed, repeat with three 50 mL washings. (Work 
rapidly to keep mat from becoming dry.) Remove filter from 
beaker and drain all H 2 from line by raising above trap level. 
Return mat and residue to beaker by breaking suction and 
blowing back. Add 200 mL boiling 1.25% NaOH and boil 
exactly 30 min. Remove beaker, and filter as above. Without 
breaking suction, wash with 25 mL boiling 1 .25% H 2 S0 4 and 
three 50 mL portions boiling H 2 0. Drain free of excess H 2 
by raising filter. Lower filter into beaker and wash with 25 mL 
alcohol. Drain line, break suction, and remove mat by blowing 
back thru filter screen into ashing dish. Proceed as in (c). 

(b) Using California buchner. — Filter contents of beaker 
thru buchner (precoated with ceramic fiber if extremely fine 
materials are being analyzed), rinse beaker with 50-75 mL 
boiling H 2 0, and wash thru buchner. Repeat with three 50 mL 
portions H 2 0, and suck dry. Remove mat and residue by snap- 
ping bottom of buchner against top while covering stem with 
thumb or forefinger and replace in beaker. Add 200 mL boiling 
1.25% NaOH and boil exactly 30 min. Remove beaker, and 
filter as above. Wash with 25 mL boiling 1.25% H 2 S0 4 , three 
50 mL portions H 2 0, and 25 mL alcohol. Remove mat and 
residue; transfer to ashing dish. 

(c) Treatment of residue. — Dry mat and residue 2 hr at 
130 ±2°. Cool in desiccator and weigh. Ignite 30 min at 
600±15°. Cool in desiccator and reweigh. 

% Crude fiber in ground sample = C 
= (Loss in wt on ignition — loss in wt of ceramic fiber blank) 

x 100/wt sample 

% Crude fiber on desired moisture basis 

- C X (100 - % moisture desired)/ 

(100 — % moisture in ground sample) 

Report results to 0,1%. 

Refs.: JAOAC 42, 222(1959); 43, 335(1960); 44, 567(1961); 
45, 578(1962); 65, 265(1982). 



978.10 Fiber (Crude) in Animal Feed 

Fritted Glass Crucible Method 

First Action 1978 
Final Action 1979 

A. Principle 

Principle is same as in 962. 09A, except sample is exposed 
to min. vac. needed to regulate filtration, and heating of sam- 
ple solns prevents gelling or pptn of possible satd solns. 

B. Apparatus and Reagents 

See reagents 962.09B(a), (b), and (f); app. 962.09C(a), (c), 
(d), and (f), and in addn: 

(a) Filtration apparatus. — System to permit application of 
min. vac. necessary for filtration and washing of each sample 
within 3-5 min. Each unit consists of reservoir manifold con- 
nected to (7) H 2 aspirator thru 120° stopcock, (2) atm. thru 
second stopcock with metering device, and (3) receptacle contg 
cone-shaped hard rubber gasket which provides vac. seal with 
crucible. Vac. gage attached to manifold indicates vac. applied 
to crucible. Crucible can be heated before and during filtration 
by flow of hot H 2 in surrounding jacket. (For photograph of 
app., see JAOAC 56, 1353(1973). Filtration unit is available 
as Model 60 1 or 602 (replacement models for Model 1 50) from 
Analytical Bio-Chemistry Laboratories, Inc., 7200 ABC Ln, 
Columbia, MO 65205). 

(b) Crucible. — Fritted glass, 50 mL, coarse porosity. Clean 
as follows: Brush, and flow hot tap H 2 into crucible to re- 
move as much ash as possible. Submerge crucible in base soln, 



82 



Animal Feed 



AOAC Official Methods of Analysis (1990) 



(c)(2), ^5 min, remove, and rinse with hot tap H 2 0. Sub- 
merge in HO (1 +1), (c) (1), >5 min, remove, and rinse tho- 
roly with hot tap H 2 followed by distd H 2 0. After 3-4 uses, 
back wash by inverting crucible on hard rubber gasket in fil- 
tration app., and flowing near-boiling H 2 thru crucible under 
partial vac. 

(c) Cleaning solns.-(J) Acid soln.— HC1 (1 + 1). (2) Base 
soln.— Dissolve 5 g Na 2 H 2 EDTA, 50 g Na 2 HP0 4 (tech. grade), 
and 200 g KOH in H 2 0, and dil. to 1 L. Storage in sep. wide 
mouth containers holding 2-3 L soln into which crucibles can 
be placed is convenient. 

C. Determination 

(Caution: See safety notes on distillation and petroleum 
ether.) 

Ext 2 g ground material with ether or pet ether (initial boil- 
ing temp., 35-38°; dry-flask end point, 52-60°; >:95% distg 
<54°, and <60% distg <40°; sp gr at 60°F, 0.630-0.660; evapn 
residue <0.002% by wt). If fat is <1%, extn may be omitted. 
Transfer to 600 mL reflux beaker, avoiding fiber contamina- 
tion from paper or brush. Add 0.25-0.5 g bumping granules, 
followed by 200 mL near-boiling 1.25% H 2 S0 4 soln in small 
stream directly on sample to aid in complete wetting of sam- 
ple. Place beakers on digestion app. at 5 min intervals and boil 
exactly 30 min, rotating beakers periodically to keep solids 
from adhering to sides. Near end of reflux ing place California 
buchner, 962.09C(d), previously fitted with No. 9 rubber 
stopper to provide vac. seal, into filtration app., and adjust 
vac. to ca 25 mm Hg (735 mm pressure). At end of refluxing, 
flow near-boiling H 2 thru funnel to warm it; then decant liq. 
thru funnel, washing solids into funnel with min. of near-boil- 
ing H 2 0. Filter to dryness, using 25 mm vac, and wash res- 
idue with four 40-50 mL portions near-boiling H 2 0, filtering 
after each washing. Do not add wash to funnel under vac; lift 
funnel from app. when adding wash. 

Wash residue from funnel into reflux beaker with near-boil- 
ing 1.25% NaOH soln. Place beakers on reflux app. at 5 min 
intervals and reflux 30 min. Near end of refluxing, turn on 
filtration app., place crucible, (b), in app., and adjust vac. to 
ca 25 mm. Flow near-boiling H 2 thru crucible to warm it. 
(Keep near-boiling H 2 flowing thru jacket during filtration 
and washing.) At end of refluxing, decant liq. thru crucible 
and wash solids into crucible with min. of near-boiling H 2 0. 
Increase vac as needed to maintain filtration rate. Wash res- 
idue once with 25-30 mL near- boiling 1 .25% H 2 S0 4 soln, and 
then with two 25-30 mL portions near-boiling H 2 0, filtering 
after each washing. (Filtering and washing takes ca 3-5 min/ 
sample.) Do not add wash to crucible under vac. 

Dry crucible with residue 2 hr at 130 ±2° or overnight at 
1 10°, cool in desiccator, and weigh. Ash 2 hr at 550 ± 10°, cool 
in desiccator, and weigh. Do not remove crucibles from fur- 
nace until temp, is <250°, as fritted disk may be damaged if 
cooled too rapidly. 

% Crude fiber = Loss in wt on ignition x 100/wt sample 

Ref.: J AOAC 61, 154(1978). 



973.18 Fiber (Acid Detergent) and Lignin 
in Animal Feed 

First Action 1973 
Final Action 1977 

(Caution: See safety notes on asbestos.) 

A Reagents 

(a) Sulfuric acid. — 72% by wt. Stdze reagent grade H 2 S0 4 
to sp gr 1.634 at 20° or 24.007V: Add 1200 g H 2 S0 4 to" 440 



mL H 2 in 1 L MCA vol. flask with cooling. Stdze to 1634 
g/L at 20° by removing soln and adding H 2 or H 2 S0 4 as 
required. 

(b) Acid-detergent soln. — Add 20 g cety.1 trimethylammon- 
ium bromide (tech. grade) to 1 L 1 .Q0N H 2 S0 4 , previously 
stdzd. Agitate to aid soln. 

(c) Asbestos. — Place 100 g asbestos in 3 L if ask contg 850 
mL H 2 0. Add 1.4 L H 2 S0 4 (tech. grade), mix, and let cool 
2 hr at room temp. Filter on large buchner and wash with H 2 0. 
Resuspend mat in H 2 and pour into bag sewn from rectangle 
of fiberglass window screening, 14 x 18 mesh (bag should be 
>45 cm wide x 30 cm deep). Wash by immersion and agi- 
tation in partly filled sink to remove fine particles. Ash re- 
covered asbestos 16 hr in 800° furnace. Store in dry form until 
use. Used asbestos may be rewashed, reashed, and reused. 
Com. prepd acid-washed asbestos is unsatisfactory unless treated 
with 72% H 2 S0 4 and ashed at 800°. 

B. Apparatus 

(a) Refluxing apparatus. — Any conventional app. suitable 
for crude fiber detns. Berzelius beakers (600 mL) and con- 
densers made from 500 mL r-b flasks are also satisfactory. 

(b) Fritted glass crucibles. — Use coarse porosity, 40-50 
mL Pyrex crucible. Wash new crucibles and ash at 500°. Re- 
move while still hot and place in 100° forced-draft oven >1 
hr. Cool 15 min in desiccator over P 2 5 or Mg(C10 4 ) 2 and 
weigh in same order samples are to be weighed. Check balance 

after each weighing if crucibles are still warm. Hold length 
of time from oven to balance pan as const as possible and 
always weigh crucibles in same order. 

C. Determination of Acid-Detergent Fiber 

Weigh 1 g air-dried sample ground to pass I mm screen, or 
approx. equiv. amt wet material, into refluxing container. Add 
100 mL acid- detergent soln at room temp. 

Heat to boiling in 5-10 min; reduce heat to avoid foaming 
as boiling begins. Reflux 60 min from onset of boiling, ad- 
justing boiling to slow, even level. Remove container, swirl, 
and filter thru weighed (W x ) fritted glass crucible, using min. 
suction. Increase vac. only as needed. Shut off vac. Break up 
filtered mat with rod and fill crucible 2 / 3 full with hot (90- 
100°) H 2 0. Stir and let soak 15-30 sec. Dry with vac. and 
repeat H 2 washing, rinsing sides of crucible. Wash twice 
similarly with acetone. 

Repeat acetone washings until no more color is removed, 
breaking up all lumps so that sol v. wets all particles of fiber. 
Remove residual acetone with vac. Dry 3 hr or overnight in 
100° forced-draft oven and weigh (W 2 ). Calc. % acid-detergent 
fiber = 100 (W 2 — W { )/S, where S = g sample x g oven- 
dried matter /g air-dried or wet matter, detd on sep. sample. 

D. Determination of Lignin 

To crucible contg fiber, 973. 18C, add 1 g asbestos. Place 
crucible in 50 mL beaker for support or arrange crucibles in 
shallow enamel pan. Cover contents of crucible with cooled 
(15°) 72% H 2 S0 4 and stir with glass rod to smooth paste, 
breaking all lumps. Fill crucible about half-way with acid and 
stir. Leave glass rod in crucible; refill with 72% H 2 S0 4 and 
stir hourly as acid drains, keeping crucible at 20-23° (cool if 
necessary). After 3 hr, filter as completely as possible with 
vac. , and wash with hot H 2 until acid-free to pH paper. Rinse 
sides of crucible and remove stirring rod. Dry crucible in 100° 
forced-draft oven, cool in desiccator over P 2 O s or Mg(C10 4 ) 2 , 
and weigh (W 3 ). Ignite crucible in 500° furnace 2 hr or until 
C-free. Place crucible while still hot into 100° forced-draft oven 

1 hr. Transfer to desiccator, cool, and weigh (W 4 ). 

Det. asbestos blank by weighing 1 g asbestos into tared cru- 
cible. Proceed as above, beginning "Cover contents of cru- 
cible ..." Record any loss in wt on ashing (W 5 ). Discontinue 



AOAC Official Methods of Analysis (1990) 



Sugars 



83 



detn of blank if asbestos blank is <0.0020 g/g asbestos. Calc. 
% acid-insol. lignin = (W 3 - W 4 - W 5 )/S. 

Refs.: JAOAC 46, 829(1963); 56, 781(1973). 

CAS-9005-53-2 (lignin) 



974.06 Sugars (Total) in Animal Feed 

Modified Fehling Solution Method 

First Action 1974 
Final Action 1975 

A. Reagents 

(a) Soxhlet modification of Fehling soln. — Prep, as in 
923.09A(a) and (b). 

(b) Invert sugar std soln. — 1.0%. Prep, as in 923.09A(c), 
but do not neutze. Dil. to 0.5% just before use for analysis of 
most products. 

(c) Lactose std soln. — 1.0%. Dissolve 5.000 g lactose in 
H 2 and dil. to 500 mL. Prep, daily. 

B. Apparatus 

(a) Lamp. — Fluorescent desk lamp or 150 watt reflector spot 
lamp, to illuminate boiling soln. 

(b) Heater. — Glas-Col mantle, 250 mL, placed over mag. 
stirrer. Adjust heat so that 50 mL H 2 contg stirring bar will 
boil in 3 min. Mag. stirring hot plate is also satisfactory. 

C. Preparation of Sample and Inversion 

(a) Feeds containing molasses .— Weigh appropriate size 
sample, prepd as in 950.02 but not ground, to provide final 
soln ca 0.5% invert sugar but ^5 g, into 250 mL P flask 
(Corning Glass Works No. 5840, or equiv.). Add 150 mL H 2 0, 
swirl to wet and mix, and heat just to bp. Let stand to cool, 
dil. to vol., mix, and let stand to settle coarse particles. Trans- 
fer 50 mL supernate to 100 mL vol. flask and add 2.5 mL HC1 
(sp gr 1.18 at 20/4°). Let stand overnight at ^25°, dil. to vol., 
and mix. (If aliquot to be used in detn is >25 mL, it is nec- 
essary to neutze inverted soln.) 

(b) Feeds containing milk products. — Weigh appropriate 
size sample to provide final soln ca 1% lactose into 250 mL 
vol. flask. Thoroly moisten sample with H 2 0, swirl to dissolve 
lactose, dil. to vol., mix, and let stand to settle coarse parti- 
cles. Proceed as in 974.06E(b). 

D. Standardization 

Fill 50 mL buret, with offset tip, with std sugar soln (invert 
sugar for use with 974.06E(a) and lactose with 974.06E(b)). 
Proceed as in 968.28D, par. 2, except use same type flask as 
used in 974. 06E, do not add H 2 0, and start stirring after addn 
of indicator. 

E. Determination 

(a) Difference method. — Add reagents and stirring bar to 
250 mL extn flask (Corning Glass Works No. 5160, or equiv.) 
or to erlenmeyer, as in 974.06D. Transfer aliquot inverted soln, 
(a), to flask so that >1 but <5 mL std soln will be required 
to reach end point, place on preheated mantle or hot plate, heat 
to bp, boil 2 min, add ca 1 mL indicator, and begin stirring. 
Complete detn by titrg with std sugar soln to same end point 
used in stdzn. Color change is not so sharp as in stdzn, but 
under suitable light it is definite, discernible, and repeatable. 

(b) Alternative method. — Fill buret with sample soln, (b), 
or inverted sample soln, (a). As in 974. 06D, place reagents 
in flask, place on heater, add sample soln to within 2 mL of 
final titrn (detd by trial), bring to bp, boil 2 min, and complete 
titrn as in (a). 



F. Calculations 

% Total sugar (as invert or lactose) 

= [(F - M) x / x 1001/IV x (lV/250) X D)] 

where F = mL std sugar required to reduce mixed Soxhlet 
reagent in stdzn; M — mL std soln required to complete detn 
(omit in alternative method); / = concn std soln; V = mL sam- 
ple soln in aliquot used; W — g sample; and D = diln factor. 
Report total sugars, expressed as invert or as lactose. 

Ref.: JAOAC57, 382(1974). 



925.05 



Sucrose in Animal Feed 
Final Action 



Place 10 g sample in 250 mL vol. flask. If material is acid, 
neutze by adding 1-3 g CaC0 3 . Add 125 mL 50% alcohol by 
vol., mix thoroly, and boil on steam bath or by partially im- 
mersing flask in H 2 bath 1 hr at 83-87°, using small funnel 
in neck of flask to condense vapor. Cool and let mixt. stand 
several hr, preferably overnight. Dil. to vol. with neut. 95% 
alcohol, mix thoroly, let settle or centrf. 15 min at 1500 rpm, 
and decant closely. Pipet 200 mL supernate into beaker and 
evap. on steam bath to 20-30 mL. Do not evap. to dryness. 
Little alcohol in residue does no harm. 

Transfer to 100 mL vol. flask and rinse beaker thoroly with 
H 2 0, adding rinsings to flask. Add enough satd neut. Pb(OAc) 2 
soln (ca 2 mL) to produce flocculent ppt, shake thoroly, and 
let stand 15 min. Dil. to vol. with H 2 0, mix thoroly, and filter 
thru dry paper. Add enough anhyd. Na 2 C0 3 or K oxalate to 
filtrate to ppt all Pb, again filter thru dry paper, and test filtrate 
with little anhyd. Na 2 C0 3 or K oxalate to make sure that all 
Pb has been removed. 

Place 50 mL prepd soln in 100 mL vol. flask, add piece of 
litmus paper, neutze with HC1, add 5 mL HC1, and let inver- 
sion proceed at room temp, as in 925.48(c). When inversion 
is complete, transfer soln to beaker, neutze with Na 2 C0 3 , re- 
turn soln to 100 mL flask, dil. to vol. with H 2 0, filter if nec- 
essary, and det. reducing sugars in 50 mL soln (representing 
2 g sample) as in 906. 03B. Calc. results as invert sugar. 

% Sucrose = [% total sugar after inversion - % reducing 
sugars before inversion (both calcd as invert sugar)] x 0.95 

Because insol. material of grain or cattle food occupies some 
space in flask as originally made up, correct by multiplying 
all results by factor 0.97, as results of large number of detns 
on various materials show av. vol. of 10 g material to be 7.5 
mL. 

Refs.: USDA Bur. Chem. Circ. 71. JAOAC 41, 276(1958); 
42, 39(1959). 

CAS-57-50-1 (sucrose) 

920.40* Starch in Animal Feed 

Final Action 
Surplus 

A. Direct Acid Hydrolysis 
See 7.080, 13th ed. 

B. Diastase Method with Subsequent Acid Hydrolysis 

See 7.067, 12th ed. 

C. Extraction with Subsequent Enzyme Hydrolysis 
See 14.075-14.080, 13th ed. 

D. In Presence of Interfering Polysaccharides 
See 22.048, 1.0th ed. 



84 



Animal Feed 



AOAC Official Methods of Analysis (1990) 



E. In Condensed or Dried Milk Products — Qualitative Test 
See 22.049, 10th ed. 

920.41* Pentosans in Animal Feed 

Final Action 
Surplus 1965 

See 22.050-22.051, 10th ed. 

920.42* Galactan in Animal Feed 

Final Action 
Surplus 1965 

See 22.052, 10th ed. 

920.43* Acidity (Water-Soluble) 

of Animal Feed 

Final Action 
Surplus 1965 

See 22.053, 10th ed. 

925.12* Mineral Salts in Animal Feed 

Final Action 
Surplus 1974 

A. Ferrous Salts 

Seel. 074, 1 2th ed. 

B. Copper Salts 

See 7.075, 12th ed. 

C. Potassium Iodide 
See 7.076, 12th ed. 

968.08 Minerals in Animal Feed 

Atomic Absorption Spectrophotometry Method 
First Action 1968 
Final Action 1969 

(Caution: See safety notes on A AS.) 

A. Apparatus 

Atomic absorption spectrophotometer. — See 965. 09A. 

B. Operating Parameters 

See Table 965.09, except use fuel-rich air-C 2 H 2 flame for 
Ca and Mg, and ranges of operation for jutg element/mL so In 
are: Ca 5-20, Cu 2-20, Fe 5-20, Mg 0.5-2.5, Mn 5-20, and 
Zn 1-5. 

C. Reagents 

(See introduction to 965. 09B. Com. prepd std solns may be 
used.) 

(a) Calcium std solns. — Prep, as in 965.09B(a). 

(b) Copper, iron, magnesium, manganese, and zinc std 
solns. — Prep, stock solns as in 965.09B(b), (c), (e), (f), and 
(g), and dil. aliquots with 0.1-0.5/V HC1 to make >4 std solns 
of each element within range of detn. 

D. Preparation of Sample Solution 

(a) Dry ashing (not applicable to mineral-mix feeds) . — Ash 
2—1 g sample in well-glazed porcelain dish. Start in cold fur- 
nace, bring to 550°, and hold 4 hr. Cool, add 10 mL 3/V HC1, 
cover with watch glass, and boil gently 10 min. Cool, filter 
into 100 mL vo\. flask, and dil. to vol. with H 2 0. Subsequent 



dilns with 0.1-0.5/V HC1 may be necessary to bring sample 
solns into anal, range, except for Ca. Final Ca diln must con- 
tain enough La soln, 965.09B(d), to provide 1% La concn 
after diln to vol. with H 2 0. 

(b) Wet digestion. — Proceed as in 935.13A(a), adding 25 
mL HN0 3 for each 2.5 g sample and dilg to 100 mL with FLO. 
Digestion can be made at low heat on hot plate, using 600 mL 
beaker covered with watch glass. Subsequent dilns with 0.1- 
0.5/V HC1 may be necessary to bring sample solns into anal, 
range, as in (a). 

E. Determination and Calculation 
See 965.09D-E. 

Refs.: JAOAC 51, 776(1968); 54, 666(1971); 59, 937(1976); 
60, 465(1977). 

CAS-7440-70-2 (calcium) 
CAS-7440-50-8 (copper) 
CAS-7439-89-6 (iron) 
CAS-7439-96-5 (manganese) 
CAS-7440-66-6 (zinc) 



927.02 Calcium in Animal Feed 

Dry Ash Method 
Final Action 

(Applicable to mineral feeds only) 

Weigh 2 g finely ground sample into Si0 2 or porcelain dish 
and ignite in furnace to C-free ash, but avoid fusing. Boil res- 
idue in 40 mL HC1 (1+3) and few drops HN0 3 . Transfer to 
250 mL vol. flask, cool, dil. to vol., and mix thoroly. Pipet 
25 mL clear liq. into beaker, dil. to ca 100 mL, and add 2 
drops Me red, 984,13B(c) Add NH 4 O.H (1 + 1) dropwise to 
pH 5.6, as shown by intermediate brownish-orange. If over- 
stepped, add HC1 (1 +3) with dropper to orange. Add 2 more 
drops HCJ (1 + 3). Color should now be pink (pH 2.5-3.0), 
not orange. Dil. to ca 150 mL, bring to boil, and slowly add, 
with const stirring, 10 mL hot satd (4.2%) soln of (NH 4 ) 2 C 2 04. 
If red changes to orange or yellow, add HO (1 + 3) dropwise 
until color again changes to pink. Let stand overnight for ppt 
to settle. Filter supernate thru quant, paper, gooch, or fritted 
glass filter (fine Pyrex is preferable), and wash ppt thoroly with 
NH4OH (1 + 50). Place paper or crucible with ppt in original 
beaker, and add mixt. of 125 mL LLO and 5 mL H 2 S0 4 . Heat 
to >70° and titr. with 0AN KMn6 4 , 940.35, to first slight 
pink. Presence of paper may cause color to fade in few sec. 
Correct for blank and calc. % Ca. 

Refs.: JAOAC 10, 177(1927); 19, 93, 574(1936); 28, 80(1945). 

CAS-7440-70-2 (calcium) 



935.13 Calcium in Animal Feed 

Wet Ash Method 
Final Action 

A. Preparation of Solution 

(Caution: See safety notes on nitric acid and perchloric acid.) 

(a) Weigh 2.5 g sample into 500 or 800 mL Kjeldahl flask. 
Add 20-30 mL HNO3 and boil gently 30-45 min to oxidize 
all easily oxidizable matter. Cool soln somewhat and add 10 
mL 70-72% HCIO4. Boil very gently, adjusting flame as nec- 
essary, until soln is colorless or nearly so and dense white 
fumes appear. Use particular care not to boil to dryness (Dan- 



AOAC Official Methods of Analysis (1990) 



Minerals 



85 



ger!) at any time. Cool slightly, add 50 mL H 2 0, and boil to 
drive out any remaining N0 2 fumes. Cool, dil., filter into 250 
mL vol. flask, dil. to vol., and mix thoroly. 

(b) Weigh 2.5 g finely ground sample into Si0 2 or porcelain 
dish and ignite as in 942.05. Add 40 mL HC1 (1 +3) and few 
drops HN0 3 to residue, boil, transfer to 250 mL vol. flask, 
cool, dil. to vol., and mix thoroly. 

B. Determination 

Pipet suitable aliquot of clear soln, 935.13A(a) or (b), into 
beaker, dil. to 100 mL, and add 2 drops Me red, 984.13B(c). 
Continue as in 927.02, beginning "Add NH 4 OH (1 + 1) drop- 
wise ..." except use 0.05 N KMn0 4 for titrn. 

(100 mL is suitable aliquot of sample soln for grain feeds; 
for mineral feeds, 25 mL aliquot may be taken and titrd with 
0. \N KMn0 4 . For suitable precision, size of sample, aliquot, 
and concn of KMn0 4 must be so adjusted that >20 mL std 
KMn0 4 soln is used.) 

Refs.: Ind. Eng. Chem. Anal. Ed. 7, 116, 167(1935). JAOAC 
30, 606(1947); 31, 98(1948); 32, 650(1949); 33, 
162(1950); 34, 563(1951). 

CAS-7440-70-2 (calcium) 



943.01 Chlorine (Soluble) 

in Animal Feed 

Titrimetric Method 
Final Action 

A Reagents 

(a) Potassium chloride std soln. — 0.001 g Cl/mL. Recrys- 
tallize reagent KC1 3 times from H 2 0, dry at 110°, and heat 
at ca 500° to const wt. Dissolve 2.1028 g in H 2 and dil. to 
1 L. 

(b) Silver nitrate soln. — Dissolve 5 g AgN0 3 in 1 L H 2 
and adjust soln so that 1 mL — 1 mL std KC1 soln. 

(c) Potassium thiocyanate soln. — Dissolve 2.5 g KSCN in 
1 L H 2 and adjust so that 1 mL = 1 mL std AgN0 3 soln. 
Stdze as in 942.36C. 

(d) Ferric sulfate soln.— Dissolve 60 g Fe 2 (S0 4 ) ? -nH 2 in 
H 2 and dil. to 1 L. 

(e) Ferric sulfate indicator. — To filtered 25% soln of 
Fe 2 (S0 4 )ynH 2 0/add equal vol. HN0 3 . 

B. Determination 

Transfer 3 g sample to 300 mL erlenmeyer. Add 50 mL 
Fe 2 (S0 4 ) 3 soln (accurately measured), swirling flask to prevent 
caking of sample and to facilitate soln of CI. Add 100 mL (also 
accurately measured) NH 4 OH (1 + 19). Swirl flask just enough 
to ensure soln of CI and thoro mixing of soln. (Very little 
swirling is necessary. If soln is agitated by vigorous vertical 
shaking, filtration will be difficult.) Let mixt. settle 10 min. 
Filter thru dry 11 cm Whatman No. 41 paper, or equiv. Use 
50 mL aliquots (V3 of total) on samples low in CI (0-2% CI) 
and 25 mL aliquots ('/e of total) on samples high in CI (>2%). 
For mineral and other feeds contg >10% CI, weigh 1 g and 
use 15 mL (V10 of total). 

If approx. % CI in sample is not known, take 10 mL aliquot 
for trial titrn. To this add 10 mL HNO a and 10 mL Fe 2 (S0 4 ) 3 
indicator. Dil. to ca 50 mL. Add 0.5 mL KSCN soln and im- 
mediately add, with stirring, enough AgN0 3 soln to entirely 
eliminate any reddish color. From this titrn calc. vol. AgN0 3 
soln necessary to ppt all CI in aliquot to be used, adding excess 
equal to ca 10% total vol. necessary, altho somewhat greater 
excess will not affect results. Use min. total of 10 mL. 

To sample aliquot in 250 mL beaker add 10 mL HN0 3 and 



10 mL Fe 2 (S0 4 ) 3 indicator (or 20 mL soln contg equal vols of 
these solns). Add, with stirring, calcd vol. AgN0 3 soln. Heat 
to boiling and cool to room temp., stirring enough to coagulate 
ppt. (Cooling may be hastened by immersion of beakers in 
cold H 2 0.) Titr. excess AgN0 3 with KSCN. End point is in- 
dicated by first appearance of reddish tint that persists 15 sec. 
For accurate work, use ref. soln contg all ingredients except 
KSCN. End point is first change in color. 

Refs.: JAOAC 26, 87(1943); 28, 80(1945). 

CAS-7782-50-5 (chlorine) 



969.10 Chlorine (Soluble) in Animal Feed 

Potentiometric Method 

First Action 1969 
Final Action 1970 

A Apparatus 

Potentiometer. — With Ag-AgCl reference electrode and Ag- 
indicating electrode (Fisher Scientific Co. No. 9-313-216 and 
13-639-122, or equiv.). 

B. Standardization 

Weigh 125 mg dry NaCl into 400 mL beaker. Add 200 mL 
H 2 and 1 mL HN0 3 . 

Null potentiometer and titr. NaCl soln with 0AN AgN0 3 
soln. Plot mL AgN0 3 soln against mv or scale readings. Add 
titrant in small enough increments so that voltage end point is 
obvious. Use same end point for samples. 

C. Determination 

(a) Samples containing less than 5% sodium chloride. — 
Weigh 5.844 g sample into 400 mL beaker. Add ca 200 mL 
H 2 and 1 mL HNO3. Swirl mixt. gently and let stand 10 min 
for complete soln of chlorides. Titr., while stirring, to same 
voltage end point as in stdzn. 

% NaCl - mL 0AN AgNO 3 /10 

(b) Samples containing more than 5% sodium chloride. — 
Weigh 5.844 g sample into 200 mL vol. flask. Add ca 190 
mL H 2 and 1 mL HN0 3 , dil. to vol. with H 2 0, mix, and let 
stand 10 min. Transfer aliquot contg equiv. of ca 125 mg NaCl 
to 400 mL beaker, dil. to ca 200 mL, add 1 mL HN0 3 , and 
titr. as in (a). 

% NaCl - diln factor x mL 0.1/V AgNO 3 /10 

Ref.: JAOAC 52, 607(1969). 

CAS-7782-50-5 (chlorine) 



952.02 



Cobalt in Animal Feed 

Colorimetric Method 

Final Action 



A. Reagents 

(a) Cobalt, std soln. — 0.05 mg Co/mL. Dissolve 0.2385 g 
CoS0 4 .7H 2 (do not dry; use as received) in H 2 and dil. to 
1 L. Dil., if necessary, to suitable concn to prep, std curve. 

(b) Nitroso-R salt soln. —Dissolve 1 gC 10 H 4 6H.NO(SO 3 Na)2 
in H 2 and dil. to 500 mL. 

(c) Spekker acid. — Mix 150 mL 85% H3PO4 and 150 mL 
H 2 S0 4 , and dil. to 1 L with H 2 0. 

(d) Sodium acetate soln. — Dissolve 500 g NaOAc.3H 2 in 
HoO and dil. to 1 L with H,0. 



86 



Animal Feed 



AOAC Official Methods of Analysis (1990) 



B. Preparation of Standard Curve 

To 1,2, etc., up to 11 mL portions std Co soln in 100 mL 
vol. flasks add 2 mL Spekker acid, 10 mL nitroso-R salt soln, 
and 10 mL NaOAc soln. Prep, blank by using 2 mL Spekker 
acid and 10 mL NaOAc soln, but omitting nitroso-R salt soln. 
Bring solns to bp on hot plate. Add 5 mL HN0 3 and boil S:l, 
but <2 min. Cool, and dil. to 100 mL. 

C. Determination 

{Caution: See safety notes on nitric acid and hydrogen 
sulfide.) 

Ash 2 g sample 2 hr at 600°, transfer to 200 mL vol. flask 
with 20 mL HC1 and 50 mL FLO, boil 5 min, cool, and dil. 
to vol. Let soln settle. Pipet suitable aliquot into small flask. 
For samples contg 0.01-0.2% Co use equiv. of 0.25 g sample. 
For other samples, take aliquot contg <0.5 mg Co. Soln no 
longer appears to follow Beer's law above this amt. 

Pass brisk current of H 2 S thru soln 10 min. Filter directly 
into 100 mL vol. flask thru Whatman No. 40 paper. Wash with 
ca 50 mL 1% H 2 S0 4 satd with H 2 S. Add 2 small glass beads 
and boil off H 2 S. (Flasks must be given individual attention, 
as violent bumping may occur.) Shake flasks often. Add 5 mL 
HN0 3 and boil until nitrous fumes no longer appear. (Take 
care, as vol. of soln will be low and bumping and spattering 
may occur. At first indication of this, remove immediately from 
hot plate.) Small amt HNO3 remaining will not affect result. 
Cool, add 2 drops phthln, and adjust to first faint pink with 
ca 30% NaOH soln. Immediately add 2 mL Spekker acid fol- 
lowed by 10 mL nitroso-R salt soln and 10 mL NaOAc soln. 
Bring to vigorous boil, carefully add 5 mL HNO3, and boil 
S:l but ^2 min. Cool, and dil. to vol. 

Compare color with std Co solns in photoelec. colorimeter, 
using green or No. 54 filter, or in spectrophtr at 540 nm. Read 
color within 2 hr. Report % Co to third decimal place. 

Ref.: JAOAC 35, 559(1952). 

CAS-7440-48-4 (cobalt) 



947.03 Copper in Animal Feed 

Colorimetric Method 
Final Action 

A. Preparation of Standard Curve 

Dissolve 1 .9645 g CuS0 4 .5H 2 in H 2 and dil. to 500 mL. 
(1 mL— 1 mg Cu.) Use from 1 to 10 mL of this soln to prep. 
set of stds in 100 mL Pyrex g-s vol. flasks. Add 4 mL HC1, 
dil. to 50 mL, add 5 mL tetraethylenepentamine , dil. to vol. 
with H 2 0, stopper, and mix thoroly. Prep, blank, using all 
reagents except Cu. Filter blank and stds before reading color 
as in 947.03B. 

B. Determination 

Prep, sample soln as in 952. 02C, using 8 g sample. Pipet 
50 mL aliquot into 100 mL Pyrex g-s vol. flask, add 5 mL 
tetraethylenepentamine, dil. to vol. with H 2 0, and mix 
thoroly. Filter, and compare colors within 30 min in photo- 
elec. colorimeter (red or No. 66 filter) or read in spectrophtr 
at 620 nm. Report % Cu to third decimal place. 

Refs.: Anal. Chem. 19, 325(1947). JAOAC 37, 246(1954); 
38, 222(1955). 



975.08 



Fluorine in Animal Feed 

First Action 1975 
Final Action 1976 

Co/orimetric Method 



A. Determination 

See 944.08, especially 944. 08E. 



Ion Seiective Electrode Method 

B. Apparatus 

(a) Electrodes. — Fluoride ion selective electrode (Model 
9409, Orion Research Inc., or equiv.) and single junction cal- 
omel ref. electrode, plastic sleeve-type (Model 90-01, Orion 
Research Inc., or equiv.). 

(b) Magnetic stirrer. — With 4 cm (I 1 //) Teflon-coated stir- 
ring bar. Use mat to insulate sample from motor heat. 

(c) pH meter. — Corning digital Model 112 (Corning Sci- 
entific Instruments, 63 North St, Medfield, MA 02052, or 
equiv.). 



C. Reagents 



(Deionized FLO may be used.) 



CAS-7440-50-8 (copper) 



(a) Sodium acetate soln. — 3M. Dissolve 408 g NaOAc. 3H 2 
with H 2 in 1 L vol. flask. When soln warms to room temp., 
dil. to vol. with H 2 0. Adjust to pH 7.0 with few drops HOAc. 

(b) Sodium citrate soln. — 1.32M. Dissolve 222 g Na ci- 
trate. 2H 2 with ca 250 mL H 2 in 1 L vol. flask. Add 28 mL 
HCIO4, dil. to vol., and mix. 

(c) Fluoride std solns. — (J) Stock soln. — 500 ppm. Ac- 
curately weigh 1. 105 g NaF (reagent grade, dried 4 hr at 100°) 
into 1 L vol. flask. Dissolve and dil. to vol. with H 2 0, and 
mix. Store in plastic bottle. (2) Intermediate soln I. — 100 ppm. 
Pipet 20 mL stock soln into 100 mL vol. flask, dil. to vol. 
with H 2 0, and mix. (3) Intermediate soln II. — 10 ppm. Pipet 
2 mL stock soln into 100 mL vol. flask, dil. to vol. with FLO, 
and mix. (4) Working solns. — Pipet 3, 5, and 10 mL inter- 
mediate soln II and 5 and 10 mL intermediate soln I into five 
100 mL vol. flasks to prep. 0.3, 0.5, 1.0, 5.0, and 10 ppm F 
working solns, resp. To each add 10.0 mL IN HC1, 25.0 mL 
NaOAc. 3H 2 soln, (a), and 25.0 mL Na citrate soln, (b). Dil. 
to vol. with H 2 and mix. 

D. Preparation of Sample 

Accurately weigh well mixed sample contg ca 400 |xg F into 
200 mL vol. flask. Pipet in 20 mL IN HC1 and stir 20 min at 
high speed on mag. stirrer. Add 50.0 mL NaOAc soln, (a), 
and 50.0 mL Na citrate soln, (b), to dissolved sample. Dil. to 
vol. with H 2 and mix. 

E. Determination 

Connect F and ref. electrodes to pH meter, place electrodes 
in low concn F soln, and warm up pH meter. Pour 50-70 mL 
std and corresponding sample solns into sep. 100 mL beakers. 
Place electrodes in each soln and while stirring with mag. stir- 
rer at const rate, read mv of std and unknown solns. Rinse and 
blot off electrodes and stirring bar between solns. Construct 
std curve on 3 cycle semilogarithmic paper. Read ppm F of 
sample soln from std curve. 

% F = ppm F x mL sample soln x 10~ 6 X 100/g sample 
Ref.: JAOAC 58, 477(1975). 
CAS-7782-41-4 (fluorine) 



AOAC Official Methods of Analysis (1990) 



Minerals 



87 



934.02* Iodine in Mineral Mixed Feeds 

Knapheide-Lamb Method 

Final Action 
Surplus 1965 

See 22.084-22.086, 10th ed. 



935.14 Iodine in Mineral Mixed Feeds 

Elmslie-Caldwell Method 
Final Action 

(Not applicable to iodized mineral feeds contg little or no org. 
matter. Caution: See safety notes on bromine.) 

Place sample contg 3-4 mg I in 200-300 mL Ni dish. Add 
ca 5 g Na 2 C0 3 , 5 mL NaOH soln (1 + 1), and 10 mL alcohol, 
taking care that entire sample is moist. Heat on steam bath to 
remove alcohol. Then dry at ca 100° to prevent spattering upon 
subsequent heating (30 min is usually enough). 

Place dish and contents in furnace heated to 500° and keep 
at that temp. 15 min. (Ignition of sample at 500° appears to 
be necessary only to carbonize any sol. org. matter that would 
be oxidized by Br-H 2 if not so treated. Temp. >500° may 
be used if necessary.) Cool, add 25 mL H 2 0, cover dish with 
watch glass, and boil gently 10 min. Filter thru 18 cm paper 
and wash with boiling H 2 0, catching filtrate and washings in 
600 mL beaker (soln should total ca 300 mL). Neutze to Me 
orange with 85% H 3 P0 4 and add 1 mL excess. 

Add excess Br-H 2 and boil soln gently until colorless, and 
then 5 min longer. Add few crystals salicylic acid and cool 
soln to ca 20°. Add 1 mL 85% H 3 P0 4 and ca 0.5 g KI, and 
titr. I with 0.0057V Na 2 S 2 3 , adding starch soln when liberated 
I color is nearly gone, f mL 0.0057V Na 2 S 2 3 = 0.1058 mg I. 

Refs.: JAOAC 18, 338(1935); 21, 596(1938); 23, 688(1940); 
33, 83(1950). 



CAS-7553-56-2 (iodine) 



917.04 Manganese (Acid-Soluble) 

in Animal Feed 
Colorimetric Method 
Final Action 

A. Reagent 

Potassium permanganate std soln. — 500 ppm Mn. Prep, and 
stdze as in 940.35, except use 1.4383 g KMn0 4 and 0.12 g 
Na oxalate. Transfer aliquot contg 20 mg Mn to beaker. Add 
100 mL H 2 0, 15 mL H 3 P0 4 , and 0.3 g Ki0 4 , and heat to bp. 
Cool, and dil. to 1 L. Protect from light. Dil. this soln contg 
20 ppm Mn with H 2 (previously boiled with 0.3 g KIO4/L) 
to make convenient working stds in range of con ens to be 
compared. 

B. Determination 

(Caution: See safety notes on nitric acid and sulfuric acid.) 

Ash weighed sample, 5—15 g, at dull red heat (ca 600°) in 
porcelain dish. Cool, and add 5 mL H 2 S0 4 and 5 mL HN0 3 
to ash in dish or to ash transferred to beaker with 20-30 mL 
H 2 0. Evap. to white fumes. If C is not completely destroyed, 
add addnl portions HN0 3 , boiling after each addn. Cool slightly, 
transfer to 50 or 100 mL vol. flask, and add vol. dil. H 3 P0 4 



soln (8 + 92) equal to Vs vol. of flask (25 or 50 mL). Cool, 
dil. to vol., mix, and filter or let stand until clear. 

If 50 mL flask was used, pipet 25 mL clear soln into beaker 
or 50 or 100 mL vol. flask and add 15 mL H 2 0. if 100 mL 
flask was used, pipet 50 mL into beaker or 100 mL flask and 
add 30 mL H 2 0. Heat nearly to bp, and with stirring or swirl- 
ing add 0.3 g KI0 4 for each 15 mg Mn present. Keep 30-60 
min at 90-100°, or until color development is complete. Cool, 
dil. to measured vol. of 50 or 100 mL, and mix. Compare 
with std KMn0 4 soln in photoelec. colorimeter or in spec- 
trophtr at 530 nm. Calc. ppm Mn. 

Refs.: J. Am. Chem. Soc. 39, 2366(1917). G. Frederick Smith 
Chemical Co. Pub. 209, 5th ed. (1950). JAOAC 22, 
78, 673(1939); 24, 865(1941); 25, 892(1942). 

CAS-7439-96-5 (manganese) 



964.06 Phosphorus in Animal Feed 

Alkalirnetric Ammonium Molybdophosphate Method 
Final Action 

A. Reagents 

(a) Molybdate soln. — Dissolve 100 g Mo0 3 in mixt. of 144 
mL NH 4 OH and 271 mL H 2 0. Cool, and slowly pour soln, 
stirring constantly, into cool mixt. of 489 mL HN0 3 and 1 148 
mL H 2 0. Keep final mixt. in warm place several days or until 
portion heated to 40° deposits no yellow ppt. Decant soln from 
any sediment and keep in g-s vessels. 

(b) A cidified molybdate soln . — To 1 00 m L mol y bdate soln, 
(a), add 5 mL HN0 3 . Filter immediately before use. 

(c) Sodium hydroxide std soln. — Dil. 324.03 mL \N alkali, 
carbonate-free, 936.16, to 1 L. (100 mL of this soln should 
neutze 32.40 mL \N acid; 1 mL = 1 mg or 1% P 2 5 on basis 
of 0.1 g sample.) (Since burets in const use may become 
so corroded as to increase their capacity, test them at least 
annually.) 

(d) Std acid soln. — Prep, soln of HC1 or of HN0 3 , corre- 
sponding to concn of (c) or to V2 this concn, and stdze by titrn 
against (c), using phthln. 

0. Determination 

Prep, sample soln as in 935.13A(a). Pipet, into beaker or 
flask, aliquot corresponding to 0.4 g sample for P 2 5 content 
of sample <5%; 0.2 g for 5-20%; 0.1 g for >20%." Add 5- 
10 mL HNO3, depending on method of soln (or equiv. in 
NH4NO3); then add NH 4 OH until ppt that forms dissolves only 
slowly on vigorous stirring, dil. to 75-100 mL, and adjust to 
25-30°. If sample does not give ppt with NH 4 OH as test of 
neutzn, make soln slightly alk. to litmus paper with NH 4 OH 
and then slightly acid with HN0 3 (1 +3). Add 20-25 mL aci- 
dified molybdate soln for P 2 5 content <5%; 30-35 mL for 
5-20%; and enough acidified molybdate soln to ensure com- 
plete pptn for >20%. Shake or stir mech. 30 min at room 
temp.; decant at once thru filter and wash ppt twice by de- 
canting with 25-30 mL portions H 2 0, agitating thoroly and 
allowing to settle. Transfer ppt to filter and wash with cold 
H 2 until filtrate from 2 fillings of filter yields pink color on 
adding phthln and I drop of the std alkali. Transfer ppt and 
filter to beaker or pptg vessel, dissolve ppt in small excess of 
the std alkali, add few drops of phthln, and titr. with std acid. 
Report as % P. 

Ref.: JAOAC 47, 420(1964). 

CAS-7723-14-0 (phosphorus) 



88 



Animal Feed 



AOAC Official Methods of Analysis (1990) 



965.17 Phosphorus in Animal Feed 

Photometric Method 

First Action 1965 
Final Action 1966 

(Not applicable to mineral -mix feeds. Dry ashing procedure is 

not applicable to feeds or mineral mixes contg monobasic Ca 

phosphate.) 

A. Apparatus 

Spectrophotometer. — Capable of isolating 400 nm band and 
accepting ^15 mm diam. cells. 

B. Reagents 

(a) Molybdovanadate reagent. — Dissolve 40 g NH 4 molyb- 
date.4H 2 in 400 mL hot H 2 and cool. Dissolve 2 g NH 4 
metavanadate in 250 mL hot H 2 and cool; add 250 mL 70% 
HC10 4 . {Caution: See safety notes on perchloric acid.) Grad- 
ually add molybdate soin to vanadate soln with stirring, and 
dil. to 2 L. 

(b) Phosphorus std solns. — (J) Stock soln. — 2 mg P/mL. 
Dissolve 8.788 g KH 2 P0 4 in H 2 and dil. to 1 L. (2)Working 
soln. — 0.1 mg P/mL. Dil. 50 mL stock soln to 1 L. 

C. Preparation of Standard Curve 

Transfer aliquots of working std soln contg 0.5, 0.8, 1.0, 
and 1.5 mg P to 100 mL vol. flasks. Treat as in 965. 17D, 
beginning "Add 20 mL molybdovanadate reagent, ..." Prep, 
std curve by plotting mg P against %T on semilog paper. 

D. Determination 

Ash 2 g sample, in 150 mL beaker, 4 hr at 600°. Cool, add 
40 mL HC1 (1+3) and several drops HN0 3 , and bring to bp. 
Cool, transfer to 200 mL vol. flask, and dil. to vol. with H 2 0. 
Filter, and place aliquot contg 0.5-1.5 mg P in 100 mL vol. 
flask. Add 20 mL molybdovanadate reagent, dil. to vol. with 
H 2 0, and mix well. Let stand 10 min; then read %T at 400 
nm against 0.5 mg std set at 100% T. (Use ^15 mm diam. 
cells.) Det. mg P from std curve. 

% P = mg P in aliquot /(g sample in aliquot X 10) 

Ref.: JAOAC 48, 654(1965); 59, 937(1976). 

CAS-7723-14-0 (phosphorus) 



964.07 Microscopy of Animal Feed 

Basic Microscopic Examination 

First Action 1964 
Final Action 1965 

4. Apparatus 

(a) Magnifier-fluorescent illuminator with desk base, 3x y 
or reading glass. 

(b) Microscopes and illuminator. — Illuminators: Illumina- 
tor for this purpose should have: compactness and flexibility; 
transformer or resistor to vary light intensity; focusing adjust- 
ment to give uniformly lighted field of view; blue-white color 
from cool low-voltage source. (I) Compound microscope . — 
For mold counting and other filth and decomposition work, 
microscope should have following min. specifications: binoc- 
ular body with inclined oculars; 4 parfocal achromatic objec- 
tives of ca 4, 10, 20, and 40 X; revolving 4-place nosepiece; 
Abbe condenser with N.A. of 1.25; 10x Huygenian or wide- 
field eyepieces; fine adjustment; mech. stage. (2) Widefield 
stereoscopic microscope recommended for filth examina- 
tion. — Microscope should have following min. specifications: 



binocular body with inclined oculars; sliding or revolving 
nosepiece to accommodate 3 objectives; 3 parfocal objectives 
1 X, 3x, and 6 or 7.5 X; paired 10 X and paired I5x widefield 
oculars; mounted on base and capable of illumination by trans- 
mitted or reflected light. 30 x is ordinarily used for routine 
examination of filter papers. Verification at higher magnifi- 
cation may be required. Following are preferred: 

(7) Widefield stereoscopic microscope . — With arm rests, 
flat stage (remove spring holders), optional substage illumi- 
nation, inclined eyepiece, and lenses to magnify ca 7-30 x, 
15 x optimum. 

(2) Compound microscope. — With mech. stage, substage 
condenser, inclined binocular eyepiece, 3 position rotating 
nosepiece, lenses to magnify ca 36-400X, 120x optimum. 

{3) Microscope illuminator. — With iris diaphragm; mova- 
ble stand holder with rod to permit adjusting light source as 
to ht and angle for substage or direct over-stage lighting; able 
to hold 2 blue glass filters or 1 blue and I ground glass; 60- 
100 watt bulb. 

(c) Sieves.— Nest of 5" No. 10, 20, 40, 60, 80, and bottom 
pan. 

(d) Stages. — Dark Co glass plates 4 X 4" (Fisher Scientific 
Co. No. 13-735); or blue paper and microscope slides. 

(e) Spot plates. — Black and white. 

(f) Forceps. — Fine pointed, curved. If necessary, bend and 
grind on emery wheel for good contact of points. 

(g) Dropping bottles. — Amber, 30 mL, as reagent 
dispensers. 

(h) Microspatula; microstirring rods made by drawing out 
glass rods; spoon. 

B. Reagents 

(a) Chloroform. — Tech. Recover by filtration and distn. 

(b) Acetone. — Tech. 

(c) Acetone, dilute. — Dil. 75 mL acetone with 25 mL H 2 0. 

(d) Dilute hydrochloric acid. — Dil. 1 vol. HC1 with 1 voL 
H 2 0. 

(e) Dilute sulfuric acid. — Dil. 1 vol. H 2 S0 4 with 1 vol. H 2 0. 

(f) Iodine soln. — Dissolve 0.75 g KI and 0.1 g I in 30 mL 
H 2 and add 0.5 mL HC1. Store in amber dropping bottle. 

(g) Millon reagent. — Dissolve, by gently warming, 1 part 
by wt Hg in 2 parts by wt HNO3. Dil. with 2 vols H 2 0. Let 
mixt. stand overnight and decant supernate. Soln contains 
Hg(N0 3 ) 2 , HgN0 3 , HNO3, and some HN0 2 . Store in g-s bot- 
tle. {Caution: See safety notes on mercury salts.) 

(h) Molybdate soln.— Add 100 mL 10% NH4NO3 soln to 
400 mL molybdate soln, 964.06A(a). Use only clear supernate 
to fill 30 mL amber dropping bottle. Discard and refill when 
crystn occurs. 

(i) Mountant I. — Dissolve 10 g chloral hydrate in 10 mL 
FLO and add 10 mL glycerol. Store in amber dropping bottle. 

(j) Mountant II. — Dissolve 160 g chloral hydrate in 100 
mL H 2 and add 10 mL HC1. 

(k) Silver nitrate soln. — 10%. Dissolve 10 g AgN0 3 in 100 
mL H 2 0. 

C. Standards 

(a) Feed ingredients. — Collect ingredients used in grain and 
stock feeds known to conform to definitions of Association of 
American Feed Control Officials as stds. Store in 4 oz bottles. 
To control insects, add ca 1 mL CS 2 , and stopper. Become 
thoroly familiar with structural appearance of stds before and 
after treatment with org. solvs. 

(b) We ed seeds. —Collect common weed seeds occurring in 
grains. Most may be found in foreign material obtained after 
sieving com. whole grains with U.S. Grain Testing Sieve hav- 
ing 5 / G4 " (2.5 mm) triangular holes. Identify from illustration 



AOAC Official Methods of Analysis (1990) 



Microscopy 



89 



in "Identification of Crop and Weed Seeds" (USDA Handbook 
219 (1963), Government Printing Office, Washington, DC 
20402). Store in numbered vials. Become familiar with those 
weed seeds designated as prohibited and restricted noxious un- 
der state laws of individual concern. (See "State Noxious-Weed 
Seed Requirements Recognized in the Administration of the 
Federal Seed Act" (USDA, Agricultural Marketing Service, 
Grain Div., Hyattsville, MD 20782).) 

Ref.: JAOAC47, 504(1964). 



970,08 Microscopy of Animal Feed 

Identification of Vegetable Tissues 
Final Action 

A. Principle 

Feeds are fractionated according to particle size and cleared 
where necessary for clear observation; conglomerates are dis- 
integrated into constituents and fractions arranged on stage 
suitable for microscopic examination at lowest magnification 
that permits identification of components when compared to 
std feed ingredients. 

B. General Methods 

(a) Scratch feeds. — Spread representative portion of sam- 
ple on white paper and examine under magnifier-fluorescent 
illuminator at 3X or with reading glass. Identify grains and 
weed seeds; note other foreign material, heat- and insect-dam- 
aged particles, live insects, and rodent excreta; examine for 
smut, ergot, and mold ("Grain Inspection Manual," USDA). 

(b) Mashes comparatively free from adhering fine parti- 
cles.— (1) Low power microscopy. — Arrange in nest form 3 
sieves that will adequately fractionate feed according to par- 
ticle size. Generally, for cattle feeds use No. 10, 20, and 40; 
for poultry feeds, No. 20, 40, and 60. Include bottom pan. 
Add ca 10 g unground feed (plastic tablespoon makes con- 
venient scoop) to nest, and sieve thoroly. With spatula, spread 
portion from each sieve on 4 x 4" Co glass stage and place 
under stereoscopic microscope. (Blue paper may also be used 
as stage.) Arrange illuminator above and near stage so light 
strikes sample at angle of ca 45° for shadow contrast. Adjust 
magnification (ca 15 X optimum), illumination, and light filters 
to individual preference for clear observation. Blue light or 
northern daylight is preferred. Examine each fraction on stage 
sep. and systematically. Observe feed particles, continually 
probing, turning, and testing resistance to pressure with for- 
ceps. Note particle size, shape, color, resistance to pressure, 
texture, odor, and major structural features. Compare with stds. 
If desired, transfer individual particles with forceps to second 
glass plate for direct comparison with corresponding tissues 
from stds. Likewise transfer and break up conglomerates by 
gentle pressure with flat end of forceps. Make list of observed 
ingredients. Neglect trace grains which may be normal inpur- 
ities in major grains. (Consult "Official Grain Standards of the 
United States," USDA, for amts of "other grains" permissible 
as impurities in whole grains.) 

(2) High power microscopy. — Lower illuminator and select 
filters so adequate blue light is reflected thru substage con- 
denser of high power microscope. With microspatula, transfer 
little of fine sievings from bottom sieve and pan to slide, add 
2 drops mountant I, stir, and disperse with microstirring rod. 
Examine microscopically (120X optimum). Compare histo- 
logically with stds. Remove slide, add 1 drop I soln, stir, and 
re-examine. Starch cells are stained pale blue to black; yeasts 
and other protein cells, pale yellow to brown. If further tissue 
clarification is desired, boil little of same fine sievings 1 min 



with ca 5 mL mountant II. Cool, transfer drop or 2 of bottom 
settlings to slide, cover, and examine microscopically. 

(c) Oily feeds or those containing large particles obscured 
by adhering fine particles, — (Most poultry feeds and un- 
knowns are best examined by this technic.) Place ca 10 g un- 
ground feed in 100 mL tall -form beaker and nearly fill with 
CHC1 3 (hood). Stir briefly and let settle ca 1 min. With spoon, 
transfer floating (org.) material to 3.5" (9 cm) watch glass, 
drain, and dry on steam bath. Sieve, and proceed as in (b). If 
desired, filter, dry, suspend fine particles in CHC1 3 , and ex- 
amine microscopically (rarely necessary). 

(d) Feeds in which molasses has caused lumpiness and oth- 
erwise obscured vision. — Place ca 10 g unground feed in 100 
mL tall-form beaker. Add 75 mL 75% acetone, stir few min 
to dissolve molasses, and let settle. Carefully decant and re- 
peat extn. Wash residue twice with acetone by decantation, 
dry on steam bath, sieve, and proceed as in (b). 

(e) Pellets or crumbles. — Gently grind few pellets at time 
in mortar with pestle with enough pressure to sep. pellet into 
its constituents, but not to break up constituents themselves. 
Sieve first grind thin No. 20 sieve and return particles re- 
maining on sieve to mortar for further grinding. Depending on 
nature of pellet, proceed with ground material as in (b), (c), 
or (d). 



970.09 Microscopy of Animal Feed 

Identification of Animal Tissues 
and Mineral Constituents 

Final Action 

A. Principle 

Feeds contg animal tissues and minerals when suspended in 
CHCI3 readily sep. into 2 fractions: (/) Org. fraction which 
floats, consisting of muscle fibers, connective tissue, dried 
ground organs, feather remains, hoof and horn particles, etc. 
from either animal or marine products, plus all vegetable tis- 
sues. (2) Mineral fraction which sinks, consisting of bones, 
fish scales, teeth, and minerals. 

B. Preparation of Sample 

Perform CHC1 3 flotation sepn as in 970.08B(c). Collect 
floating material and dry on steam bath. Decant CHCI 3 , collect 
mineral fraction, and dry on steam bath. 

C. Identification of Animal Tissue 

Examine dried floating material as in 970.08B(b). 

D. Identification of Major Mineral Constituents 

Place dried mineral fraction on nest of No. 40, 60, and 80 
sieves and bottom pan. Sieve and place the 4 fractions in sep. 
groups on same Co glass plate or blue paper stage. Examine 
under stereoscopic microscope at ca 1 5 x . Animal and fish 
bones, fish scales, and mollusc shells are generally recogniz- 
able. Salt usually occurs in cubes which may be dyed. Calcite 
form of limestone occurs as rhombohedrons. 

£ Confirmatory Tests 

With forceps, place unknown particle on glass plate and break 
up by applying gentle pressure with flat surface. Working un- 
der stereoscopic microscope, sep. particles ca 2.5 cm and place 
beside each a fractional drop of reagent solns listed by touch- 
ing end of dropper to plate. Push particle into liq. with mi- 
crostirring rod and observe what occurs at interface. Follow 
order given until pos. identification is obtained. If preferred, 
perform tests in black spot plate. 

(a) Silver nitrate soln. — (/) Crystal immediately turns chalk 
white and slowly expands: chloride, probably salt. (2) Crystal 



90 



Animal Feed 



AOAC Official Methods of Analysis (1990) 



turns yellow and yellow needles begin to grow; mono- or di- 
basic phosphate, generally dicalcium phosphate. (3) Sparingly 
sol. white needles form (Ag 2 S0 4 ): sulfate, Mn-MgS0 4 . (4) 
Particles slowly darken: bone. 

(b) Dilute hydrochloric acid, — (/) Vigorous effervescence: 
CaC0 3 . (2) Mild effervescence or none: make following tests. 

(c) Molybdate soln. — Formation of minute yellow crystals 
at some distance from particle: tricalcium phosphate, either bone 
or rock phosphate. (All phosphates react, but mono- and di- 
basic phosphates have been identified with AgN0 3 .) 

(d) Milton reagent. — (/) Disintegrated particles mostly float, 
turn pink to red (protein), and fade in ca 5 min: bone phos- 
phate. (2) Particles appear to swell and disintegrate but remain 
on bottom: defluorinated rock phosphate. (3) Particles merely 
disintegrate slowly: rock phosphate. 

(e) Dilute sulfuric acid. — Long, thin white needles slowly 
form on addn of drop of H 2 S0 4 (J + 1) to HC1 (1 + 1) soln of 
particle: confirms Ca. 



975.09 Identification of Furazolidone, 

Tylosin, and Zoalene 



See 973.80. 



963.07 Ethoxyquin in Animal Feed 

Fluorometric Method 

First Action 1963 
Final Action 1964 

A. Reagents and Apparatus 

(a) Quinine sulfate reference soln. — 1 p,g/mL 0.1 TV H 2 S0 4 . 
Dissolve 0.100 g quinine sulfate USP (dried at 120° for 3 
hr before using) in 1 L 0.LV H 2 S0 4 . Dil. 10 mL aliquot of 
this soln to 1 L with 0.17V H 2 S0 4 . Use to calibrate photoflu- 
orometer. 

(b) Ethoxyquin std solns. — Add 100.0 mg liq. ethoxyquin 
to 100 mL vol. flask and dil. to vol. with pet ether (Soln A). 
Dil. 5 mL Soln A to 100 mL with pet ether (Soln B, 50 \xg/ 
mL). Dil. 5 mL Soln B to 100 mL with pet ether (Soln C, 2.5 
pg/mL). Dil. 10 mL Soln C to 20 mL with pet ether (1.25 
|jLg/mL) and 5 mL to 25 mL (0.50 |xg/mL). 

(c) Photofluorometer.— Equipped with primary filter pass- 
ing 365 nm Hg line (Corning Glass Works No. 5874 (CS7- 
39), or equiv.) and secondary filter passing 420-500 nm 
(Corning Glass Works 3389 + 5543 + 4784, half stock thick- 
ness, or equiv.). 

B. Preparation of Standard Curve 

Adjust photofluorometer to read with pet ether and 100 
with quinine sulfate ref. soln. Obtain fluorescence readings for 
ethoxyquin std solns contg 0-2.5 (xg/mL. Plot readings against 
(Jig ethoxyquin/mL on linear paper. 



C. Determination 

Place 10 ± 0.1 g finely ground sample in 100 mL beaker 
and slurry with 50 mL MeOH. Stir and let stand 10 min. De- 
cant thru plug of glass wool into 250 mL vol. flask. Reslurry 
residue with two 50 mL portions MeOH, decant, and filter, 
combining all filtrates. Dil. to vol. with MeOH. Transfer 25 
mL aliquot to 250 mL separator, add 100 mL H 2 0, and mix 
well. Add 50 mL pet ether, stopper, and shake moderately 1 
min. Let stand few min to sep. (If emulsion forms, add ca 100 
mg NaCl crystals. After emulsion breaks, drain aq. lower layer 
into 250 mL beaker.) Transfer pet ether layer to second 250 
mL separator, return aq. layer to first separator, and re-ext 
with two 25 mL portions pet ether. 

Add 50 mL H 2 to combined pet ether exts in separator, 
stopper, and shake moderately. Let sep., drain lower aq. layer, 
and discard. Transfer pet ether layer to 100 mL vol. flask, and 
dil. to vol. with pet ether. Adjust photofluorometer as above 
and det. fluorescence readings. Obtain pg ethoxyquin/mL from 
std curve. Ppm ethoxyquin = 100 X jjig/mL. 

Add 50 mL H 2 to combined pet ether exts in separator, 
stopper, and shake moderately. Let sep., drain lower aq. layer, 
and discard, Transfer pet ether layer to 100 mL vol. flask, and 
dil. to vol. with pet ether. Adjust photofluorometer as above 
and det. fluorescence readings. Obtain pg ethoxyquin /mL from 
std curve. Ppm ethoxyquin = 100 X pg/mL. 

If untreated feed is available, prep, std curve from series of 
samples contg 0-250 pg ethoxyquin/ 10 g and carried thru detn. 

Refs.: JAOAC 44, 560(1961); 46, 306(1963); 47, 512(1964). 

CAS-9 1-53-2 (ethoxyquin) 



970.10 



See 936.11. 



Cyanogenetic Glycosides 
In Animal Feed 



970.11 Hydrocyanic Acid in Animal Feed 
See 915.03. 

Drugs in Animal Feed 

See chapter on drugs in animal feed. 

Molasses and Molasses Products in Animal Feed 

See chapter on sugars and sugar products. 



5. Drugs in Feeds 



Hussein S. Ragheb and Robert L Small idge, Associate Chapter Editors 
Purdue University 



(Medicated feeds may deteriorate under improper storage con- 
ditions. When possible, use reasonably fresh samples, store 
them in the cold, and grind just before analysis.) 

965.16 Sampling of Animal Feed 

Procedure 

Use slotted single or double tube, or slotted tube and rod, 
all with pointed ends. 

Take ^500 g sample, 1 kg preferred, as follows: Lay bag 
horizontally and remove core diagonally from end to end. Det. 
number of cores as follows: From lots of 1 — 10 bags, sample 
all bags; from lot of >: 1 1 , sample 10 bags. Take I core from 
each bag sampled, except that for lots of 1-4 bags take enough 
diagonal cores from each bag to total >5 cores. For bulk feeds 
draw >10 cores from different regions; in sampling small con- 
tainers (<J0 lb) 1 package is enough. Reduce composite sam- 
ple to amt required, preferably by riffling, or by mixing tho- 
roly on clean oil-cloth or paper and quartering. Place sample 
in air-tight container. 

A sample from less than these numbers of bags may be de- 
clared an official sample if guarantor agrees. For samples that 
cannot be representatively taken with probe described, use other 
sampling means. 



950.02 



Animal Feed 

Preparation of Sample 

Final Action 



Grind sample to pass sieve with circular openings 1 mm 
(V25") diam. and mix thoroly. If sample cannot be ground, 
reduce to as fine condition as possible. Do not grind molasses 
feeds. 

Refs.: JAOAC 33, 424(1950); 41, 223(1958); 48, 658(1965). 



963.32* 2-Acetylamino-5-Nitrothiazole 

in Feeds 
Spectrophotometric Method 

First Action 1963 

Final Action 1964 

Surplus 1977 

See 42.011-42.016, 13th ed. 

969.53 Aklomide in Feeds 

Spectrophotometric Method 

First Action 1969 
Final Action 1971 

(Applicable in presence of sulfanitran or roxarsone) 
A. Reagents 

(a) Titanous chloride soln. — 4% aq. Prep, fresh on day of 
use from 20% soln or solid TiCl 3 . 

(b) Sodium nitrite soln. — 0.1% aq. Prep, fresh on day of 
use. 



(c) Ammonium suljamate soln. — 0.5%. Dissolve 500 mg 
NH4SO3NH2 in H 2 and dil. to 100 111L. Prep, fresh weekly. 

(d) Coupling reagent. — 0.1% aq. N-naphthylethylenedi- 
amine.2HCl. Prep, fresh weekly and store in dark glass bottle 
in refrigerator. 

(e) Aklomide std solns. — 2-Chloro-4-nitrobenzamide, pu- 
rified for std use, available from Salsbury Laboratories, 2000 
Rockford Rd, Charles City, 1A 50616. (1) Stock soln. — I mg/ 
mL. Transfer 100 mg aklomide to 100 mL vol. flask, dissolve 
in ca 75 mL MeOH, dil. to vol. with MeOH, and mix well. 
(2) Intermediate soln. — 10 |xg/mL. Pipet 10 mL stock soln 
into 100 mL vol. flask, dil. to vol. with MeOH, and mix. Pipet 
5 mL into 50 mL vol. flask. Evap. to dryness on H 2 bath 
with aid of gentle air stream and cool to room temp. Add ca 
30 mL 0.X5N HC1, shake 10 min intermittently, dil. to vol. 
with 0.157V HC1, and mix well. (3) Working solns.— 0, 0.4, 
0.8, 1.2, 1.6, and 2.0 M-g/rnL. Transfer 0, 1,2, 3, 4, and 5 
mL intermediate soln to sep. 25 mL vol. flasks and dil. to vol. 
with 0.157V HC1. 

B. Preparation of Standard Curve 

Transfer 4 mL aliquot from each working std soln to sep. 
colorimetric tubes and proceed with reduction, color devel- 
opment, and measurement as in 969. 53C. Tubes contain 0, 
1.6, 3.2, 4.8, 6.4, and 8.0 |xg aklomide/tube, equiv. to 0, 
0.008, 0.016, 0.024, 0.032, and 0.040% aklomide in feed when 
5 g sample is taken. Plot A against % aklomide. 

C. Determination 

Weigh 5 g sample contg ca 0.025% aklomide into 100 mL 
vol. flask, add 75 mL MeOH, and heat 30 min in 60° H 2 
bath, shaking occasionally. Remove flask, cool to room temp., 
and dil. to vol. with MeOH. Mix thoroly and Jet stand 40 min 
to settle feed particles. 

Pipet 5 mL clear supernate into 50 mL vol. flask and dil. 
to vol. with 0.157V HCI. Mix well and filter thru Whatman 
No. 4 paper into 125 mL erlenmeyer. (If filtrate is cloudy, 
re filter.) Pipet 4 mL filtrate into each of 2 tubes, add 2 drops 
4% TiCl 3 from dropper, mix, and let stand 2 min. Add 2 drops 
107V NaOH from dropper, mix until white ppt persists, and 
acidify with 2.0 mL HCI. Mix and let stand until soln clears. 
Add 0.5 mL NaN0 2 to one tube and 0.5 mL H 2 to second 
tube as blank; mix. After 3 min, add 0.5 mL 0.5% NH 4 sul- 
famate to each tube and mix. After 2 min, add 0.5 mL cou- 
pling reagent to each, mix, and let color develop 15 min. Read 
A of soln at 545 nm in colorimeter or spectrophtr. Subtract 
reading of feed blank. Det. % aklomide in feed directly from 
std curve. 

Ref.: JAOAC 52, 438(1969). 
CAS-301 1-89-0 (aklomide) 

969.54 Aklomide in Feeds 

Thin Layer Chromatographic Qualitative Test 
Final Action 1971 
A. Reagents 

(a) Spray reagent. — Dissolve and dil. 0.1 g p-(dimethyl- 
amino)cinnamaldehyde (DMC) (No. J436, J.T. Baker, Inc.) 



91 



92 



Drugs in Feeds 



AOAC Official Methods of Analysis (1990) 



to 100 mL with 1.07V HCL (Soln is stable >1 month.) Just 
before use, add 1 mL 20% T\C\ 3 to 25 mL DMC soln and 
mix. Discard after 1 hr. 

(b) Aklomide reference std. — 1 mg/mL. See 969.53A(e)(y). 

B. Test 

Ext 10 g sample with 25 mL MeOH, shaking occasionally 
during 15 min. Filter thru Whatman No. 4 paper into 50 mL 
beaker. Cone, filtrate to ca 2 mL on steam bath. Spot ca 10 
jxL on 250 |xm silica gel G TLC plate along with ref. std and 
develop ca 30 min, in ether. Remove from tank and air dry 
ca 15 min. Spray plate with DMC-TiCl 3 reagent. (Caution: 
See safety notes on spraying chromatograms.) Aklomide forms 
reddish pink spot. Compare R f value to that of ref. std. 

Ref.: JAOAC 52, 438(1969). 

CAS-301 1-89-0 (aklomide) 



964.28 p-Aminobenzoic Acid In Feeds 

Spectrophotometric Method 
Final Action 1965 

A. Preparation of Standard Solution 

Transfer 0.100 g /?-aminobenzoic acid (99 + % purity, avail- 
able from ICN Pharmaceuticals inc., Life Sciences Group) to 
100 mL vol. flask, dissolve in 5 mL LV NaOH, and dil. to 
vol. with H 2 0. Dil. 5 mL aliquot to 200 mL with H 2 (1 mL 
= 25 jjug). Place 2, 4, and 6 mL aliquots dild soln (50, 100, 
150 (xg) in 100 mL vol. flasks, add 3 mL HC1 to each, dil. 
to vol. with H 2 0, and mix. 

B. Determination 

Transfer 5 g freshly ground feed to 250 mL vol. flask, add 
135 mL H 2 0, making slurry of first 10 mL to wet sample com- 
pletely, and then add 15 mL HC1. Mix, and place on steam 
bath 25 min, swirling occasionally until soln darkens. Cool, 
dil. to 250 mL with H 2 0, and let feed particles settle. Pipet 
50 mL into 100 mL vol. flask, dil. to vol. with H 2 0, and mix 
thoroly. Pour soln into 250 mL beaker, add filter-aid, and filter 
thru 18.5 cm Whatman No. 2 paper, or equiv., discarding first 
10-15 mL, if turbid. 

Pipet two 10 mL aliquots into 50 mL beakers, add 5 mL 
H 2 and 2 mL fresh 0.10% NaN0 2 soln, mix, and let stand 
3 min. Add 2 mL 0.50% NH 4 sulfamate soln, mix, and let 
stand 2 min. Then add, to one beaker only, 1 mL coupling 
reagent, 969.53A(d), and to other 1 mL H 2 0. Mix solns and 
wait 10 min. Det. A against H 2 at 545 nm in spectrophtr. 
(Avoid false readings due to N bubbles on cell walls.) Subtract 
blank A from sample A and calc. |xg found by ref. to std curve. 

% /?-aminobenzoic acid in feed = fxg found/ 1000 
K /?-aminobenzoate = p-aminobenzoic acid x 1.278 

Prep, stds by treating 10 mL aliquots of 3 final std solns, 
representing 5, 10, and 15 fig, as in detn, beginning ". . . 
add 5 mL H 2 ..." Plot A at 5, 10, and \5 juug and draw 
straight line. 

C. Qualitative Tests 

(To differentiate p-aminobenzoic acid, arsanilic acid, and 
sulfaquinoxaline) 

Place 10 mL prepd sample filtrate in separator. Ext with 10 
mL peroxide-free ether by vigorous shaking 30 sec. Let layers 
sep., and drain aq. layer into another separator. Re-ext with 
10 mL ether and drain aq. layer into third separator for third 
extn with same vol. ether. After final extn, drain aq. layer into 
fourth separator, add 5 mL H 2 0, mix, and couple soln as in 
964.28B, second par. Wait 10 min, add 5 drops HC1 and 10 



mL isoamyl alcohol, and ext gently ca 30 sec. Let stand until 
layers sep. Red color in sol v. is due to p-aminobenzoic acid; 
that in lower layer, to arsanilic acid. Drain as much aq. layer 
as possible and again ext with 10 mL sol v. Arsanilic acid re- 
mains as distinct color in aq. layer, not as mere trace due to 
incomplete removal of jP-aminobenzoic acid. Combine ether 
exts, wash with 5 mL H 2 ? discard, and ext with 10 mL 1% 
Na 2 CO ? soln; acidify, and couple again to prove presence of 
sulfaquinoxaline. 

Ref.: JAOAC 47, 214(1964). 

CAS- 150- 13-0 (p-aminobenzoic acid) 
CAS-98-50-0 (arsanilic acid) 
CAS-59-40-5 (sulfaquinoxaline) 

953.1 9* 2-Amino-5-Nitrothiazole 

In Feeds 

Spectrophotometric Method 

Final Action 

Surplus 1980 

See 42.025-42.027, 13th ed. 

961.24 Amprolium in Feeds 

Spectrophotometric Method 

First Action 1961 
Final Action 1962 

A. Principle 

Amprolium is extd from feed with aq. MeOH. Ext is pu- 
rified by chromatgy on alumina, and amprolium reacts with 
2,7-naphthalenediol, K 3 Fe(CN) 6 , KCN, and NaOH in MeOH 
to form colored compd with absorption max. at 530 nm. There 
is no interference from usual components of com. feeds, vi- 
tamins, antibiotics, picolines, or pyrimidines. Nithiazide, En- 
heptin A, and nitrofurazone show some interference. 

B. Reagents 

(Caution: See safety notes on cyanides.) 

(a) Alcoholic sodium hydroxide soln. — Dil. 15.0 mL aq. 
NaOH soln, (i), with anhyd. MeOH to 200 mL. Stopper, and 
mix well. 

(b) Alumina. — Reagent grade suitable for chromatgy. Should 
pass following test: Vigorously shake 10 g alumina with 100 
mL H 2 in 250 mL g-s flask >2 min. Let settle, decant, and 
det. pH potentiometrically. pH should be 9.5-10.5. Recovery 
of amprolium may vary among different brands of alumina. 
Test column recovery by spiking ext from nonmedicated feed. 

(c) Amprolium std soln. — 25 p,g/mL. Weigh 25.0 mg Am- 
prolium Ref. Std (available from Merck & Co.) into 50 mL 
vol. flask, dissolve in dil. MeOH, (e), dil. to vol., and mix. 
Dil. 5 mL to 100 mL in vol. flask with dil. MeOH. Soln is 
stable 1 week. 

(d) Color developing reagent. — Add 5 mL K 3 Fe(CN) 6 soln 
to 90 mL naphthalenediol soln, (f ), in 250 mL g-s flask, and 
mix well. Add 5 mL KCN soln, (g), stopper, mix well, and 
let stand 30-35 min. Add 100 mL ale. NaOH soln, (a), and 
mix. Use within 75 min, filtering thru medium porosity fritted 
glass filter just before use. 

(e) Dilute methyl alcohol, — Mix 2 vols anhyd. MeOH with 
1 vol. H 2 0. Cool to room temp, before use. 

(f) Naphthalenediol soln. — Dissolve 25 mg 2,7-naphtha- 
lenediol (Eastman Kodak Co.) in 1 L anhyd. MeOH. 

(g) Potassium cyanide soln. — Dissolve 1.0 g KCN in 100 
mL H 2 0. Kept tightly stoppered, soln is stable 2 weeks. 

(h) Potassium ferricyanide soln. —Dissolve 200 mg 



AOAC Official Methods of Analysis (1990) 



Amprolium 



93 



K 3 Fe(CN) 6 in 100 mL H 2 0. Kept tightly stoppered, soln is 
stable 2 weeks. 

(i) Sodium hydroxide soln. — Dissolve 2.25 g NaOH in 200 
mL H 2 0. 

C. Extraction 

Accurately weigh amt ground feed (<15 g) contg 1.5-2.5 
mg amprolium and transfer to 250 mL g-s flask. Add 100.0 
mL dil. MeOH, stopper, and stir mag. or shake mech. 60 min. 
Filter thru Whatman No. 42, or equiv., paper and collect 25- 
40 mL clear filtrate, rejecting first 10-15 mL. Filtrate should 
be clear. Refilter, if necessary, thru fresh paper or centrf. until 
clear. 

D. Chromatography 

(a) Preparation of alumina. — To 200 g Al 2 3 , (b), add 1 
L H 2 0. Stir mixt. 30 min. Filter slurry thru fast paper on buch- 
ner funnel. Wash A1 2 3 on filter with three 100 mL portions 
of anhyd. MeOH. Air-dry under vac. until A1 2 3 reaches room 
temp. Prepd A1 2 3 should be free-flowing. Store in g-s bottle. 

(b) Preparation of column. — Constrict end of 40 cm length 
of 9- 10 mm id glass tubing by rotating in hot flame until open- 
ing is 4—5 mm. Insert small plug of Pyrex glass wool in lower 
end of tube and compress with glass rod to thickness ca 2-3 
mm. Transfer 5.0 g prepd alumina to dry tube and pack by 
gentle tapping of tube. Prep. sep. column for each sample. 
Note: Column recovery of amprolium may vary among dif- 
ferent brands of basic alumina. Test column recovery by spik- 
ing ext from nonmedicated feed. 

(c) Chromatography of feed extract.— Pipet 25 mL clear 
ext onto column and let pass thru column by gravity. Reject 
first 3 mL eluate and collect next 5 mL for color development. 

E. Determination 

Mark 3 sep. 15 mL centrf. tubes as X, S, and B. To X add 
4.00 mL clear eluate from column; to S add 4.00 mL ampro- 
lium std soln, and to B add 4.00 mL dil. MeOH as blank. Add 
10.0 mL color developing reagent to each tube, stopper, mix, 
and let stand 20 min. Centrf. 2-3 min, decant into 1 cm cells, 
and cover. (If solns are not clear and free from suspended par- 
ticles, decant into cells thru small plug of Pyrex glass wool.) 
Det. A of solns X and S in spectrophtr or colorimeter at 530 
nm against soln B as ref. within 20-25 min after adding color 
developing reagent. 

% Amprolium in feed - (2.5A x C)/(A' x W) 

where A and A' refer to sample and std, resp., C = mg am- 
prolium in final aliquot of std soln (0.100 mg), and W = g 
original sample. 

Refs.: JAOAC 44, 5(1961); 62, 399(1979); 72, 105(1989). 

CAS- 121-25-5 (amprolium) 

965.47 Amprolium in Feeds 

Fluorometric Method 

First Action 1965 
Final Action 1967 

(Applicable in absence of antibiotics except procaine penicillin 
and chlortetracycline) 

A. Reagents 

(a) Amprolium std solns. — (7) Stock soln. — 0.20 mg/mL. 
Weigh 20.0 mg Amprolium Ref. Std (available from Merck 
& Co.) and dissolve in enough TCA soln, (d), to make 100,0 
mL. (2) Working soln. — 1 ^g/mL. Dil. 5.00 mL stock soln 
to 100 mL with TCA soln and mix well. Further dil. 10 mL 
of this soln to 100 mL with H 2 0, and mix well. 



(b) Potassium ferricyanide soln. — Dissolve 2 g K 3 Fe(CN) 6 
in 100 mL H 2 0.' 

(c) Silver nitrate soln. — Dissolve 5 g AgN0 3 in 100 mL 
H 2 0. 

(d) Trichloroacetic acid (TCA) soln. — Dissolve 5 g 
CCI3COOH in 100 mL H 2 0. 

B. Extraction 

Grind feed sample to pass No. 20 sieve and mix thoroly. 
(High-speed blender grinds most feeds to desired fineness in 
ca 3 min.) Weigh sample contg ca 750 jmg amprolium and 
transfer to 250 mL g-s flask. Add 100.0 mL TCA soln, stop- 
per, and agitate 30 min on mag. stirrer or mech. shaker. 

Filter by gravity thru Whatman No. 42 paper, rejecting first 
5 mL. Collect ^10 mL clear filtrate. Transfer 5.00 mL clear 
ext to 50 mL vol. flask, dil. to vol. with H 2 0, and mix well. 
This is dild sample ext. 

C. Development of Fluorophor 

Mark three 50 mL centrf. tubes X, Y> and Z. To tube X add 
15.00 mL dild sample ext; to tube Y add 1.50 mL TCA soln 
and 13.50 mL H 2 as blank soln; and to tube Z add 15.00 mL 
amprolium working std soln. To all tubes add 5.00 mL NaOH 
soln (3 + 10), stopper with polyethylene stoppers, and mix 
well. Immediately add 0.50 mL AgN0 3 soln to all tubes, stop- 
per, and mix well. Let all tubes stand 2 min. Then to all tubes 
add 3.0 mL K 3 Fe(CN) 6 soln, stopper, mix, and let stand 3.0 
min. 

During this 3 min wait, add 15 mL /7-BuOH to all tubes, as 
overlay, and stopper. After 3 min, vigorously shake all tubes 
1 .0 min, and centrf. 1 min. Transfer 10.0 mL aliquots of upper 
BuOH layer from all tubes to test tubes. Add 1 .00 mL absolute 
alcohol to each tube and mix well. 

D. Measurement of Fluorescence 

(Caution: See safety notes on photofluoro meters.) 

(a) For instruments designed to accommodate 10 X 10 mm 
cells and using monochromatic light for excitation. — Set ac- 
tivation wavelength at 400 nm (uncorrected) and emission 
wavelength at 455 nm (uncorrected). Transfer ca 2.0 mL fluo- 
rophor BuOH ext to cell and read. 

(b) For instruments designed to accommodate JO x 40 mm 
cells and using filters to adjust wavelengths for excitation and 
emission. — Use Kopp Glass Co. No. C5840 filter placed after 
light source to adjust excitation wavelength and Kopp No. C3385 
filter placed behind cell to adjust emission wavelength. Trans- 
fer entire contents of test tube contg extd fluorophor to cell 
and read. 

E. Calculations 

% Amprolium in feed 

- (X - Y) x C/[150 x (Z - Y) x W] 

where X, Y, and Z are fluorescence readings of sample, re- 
agent blank, and std, resp.; C = \xg in 15 mL std soln (15.0); 
and W - g sample. 

Ref.: JAOAC48, 285(1965). 

CAS-121-25-5 (amprolium) 



981.27* Arprinocid in Feeds 

Liquid Chromatographic Method 

First Action 1981 
Surplus 1988 



See 42.021-42.026, 14th ed. 



94 



Drugs in Feeds 



AOAC Official Methods of Analysis (1990) 



982.42* Arprinocid in Premixes 

Spectrophotometric Method 

First Action 
Surplus 1988 

See 42.027-42.032, 14th ed. 



954.17 Arsanilic Acid in Feeds 

Spectrophotometric Method 
Final Action 1960 

(Applicable in absence of sulfonamides) 

A. Determination 

Transfer 4.0 g freshly ground sample to 200 mL vol. flask, 
and add ca 80 mL H 2 and 4 mL 0.5N NaOH. Place flask on 
steam bath ca 5 min, swirling occasionally. Carefully add 20 
mL HC.I, mix, and cool to room temp. Dil. to vol. with H 2 0, 
mix, pour into 250 mL beaker, add some Filter-Cel, or equiv. , 
and filter thru Whatman No. 42, or equiv., paper, discarding 
first 5 mL. 

Pipet 5 mL aliquots of clear filtrate into each of two 20 X 
175 mm test tubes. To each tube add 2 mL 0.1% NaN0 2 soln, 
mix, and let stand 5 min. Add 2 mL 0.5% NH 4 sulfamate soln 
and let stand 2 min. Then add, to 1 tube only, 1 mL coupling 
reagent, 969.53A(d), mix, and let stand 10 min before dilg 
both solns to vol. of 15 mL. Mix well (if bubbles appear, filter 
thru glass wool), and det. A against H 2 at 538 nm in spec- 
trophtr or with 540 nm filter in photometer. Subtract A of blank 
from sample A. Det. jxg arsanilic acid in aliquot (equiv. to 100 
mg sample) from std curve. 

0. Preparation of Standard Curve 

Transfer 0.100 g pure arsanilic acid to 100 mL vol. flask, 
add ca 20 mL H 2 and 2 mL 0.5N NaOH, and dissolve. Dil. 
to vol. with H 2 and mix well. Transfer 10 mL to 100 mL 
vol. flask, dil. to vol. with H 2 0, and mix well. Dil. 5 mL of 
this soln to 250 mL with H 2 in vol. flask, and mix well (1 
mL — 2 |xg arsanilic acid). Pipet aliquots of 0, 2, 3, 5, and 
8 mL of this std soln into 20 x 175 mm test tubes, add 1 mL 
HO (1 + 1) to each tube, and continue as in 954. 17A, be- 
ginning "To each tube add 2 mL 0.1% NaN0 2 ..." Subtract 
blank A from A of stds and plot differences against 4, 6, 10, 
and 16 |xg arsanilic acid in aliquots. 

Refs.: JAOAC 37, 257(1954); 40, 452(1957). 

CAS-98-50-0 (arsanilic acid) 



957.22 Arsenic (Total) in Feeds 

Colorimetric Test 

First Action 1957 
Final Action 1960 

A. Reagents 

(a) Arsenic trioxide. — NIST As 2 3 SRM 83, or equiv. 

(b) Magnesium oxide-magnesium nitrate slurry. — Suspend 
75 g MgO and 105 g Mg(N0 3 ) 2 .6H 2 in enough H 2 to make 
1 L. Agitate vigorously before addn to sample. (Freshly prepd 
slurry gives ash which is easily disturbed by air currents.) 

(c) Stannous chloride soln. — Dissolve 40 g As-free SnCl 2 - 
H 2 in HC1 and dil. to 100 mL with HC1. Effective as long 
as it discharges yellow color in sample ext. 

(d) Absorbing soln. — Transfer with graduate 25 mL 1.5% 
HgCl 2 soln, and with pipet 3.75 mL 6/V H 2 S0 4 and 3.75 mL 



0.03/V KMn0 4 , into 250 mL graduate. Dil. to 250 mL with 
H 2 and mix. Prep, fresh daily. 

(e) Ammonium molybdate reagent. — Dissolve 1 g 
(NH4) 2 Mo0 4 in 100 mL 5 AN H 2 S0 4 . Soln keeps several weeks. 
(Prep. 5.4V H 2 S0 4 by dilg 6N (9 + 1).) 

(f) Hydrazine sulfate reagent. — 0.15%. Dissolve 0.15 g 
N2H4.H2SO4 in 100 mL H 2 0. Soln keeps several weeks. 

B. Apparatus 

(Do not clean app. and glassware with detergents, as they in- 
terfere with color development. Haemo-Sol, available from 
Scientific Products, Inc., or equiv., is satisfactory.) 

(a) Evaporating dishes. — 70 mL; Coors No. 430, size 00A, 
or equiv. 

(b) Arsine evolution apparatus .—Bend 6 mm id glass tub- 
ing at 120 3 angle ca 10 cm from one end and at 60° angle ca 
15 cm from other end. Plug shorter end with glass wool im- 
pregnated with satd Pb(OAc) 2 soln and insert in rubber stop- 
per, placed in top of 125 mL erlenmeyer, so that end of tube 
projects just below stopper. Plug other end with un impreg- 
nated glass wool and connect thru rubber tubing to glass tube, 
constricted at lower end, that reaches to bottom of 50 mL large 
neck vol. flask, or if preferred, 50 mL centrf. tube, marked 
exactly at 50 mL and approx. at 20 mL. 

C. Preparation of Sample Solution 

Weigh ground sample contg <50 |xg As (unless aliquot is 
to be taken from digested soln) into 70 mL ashing dish. If 
>2.5 g sample is used, increase amt of slurry and size of ash- 
ing dish. Add ca 10 mL well mixed slurry, (b), and enough 
H 2 to permit thoro mixing with stirring rod. Rinse stirring 
rod, and dry sample at 100°. Ash 2-4 hr at 550-600°. (Slight 
C residue does not interfere. Use care to avoid loss of ash.) 

Cool, and moisten residue with H 2 0. Cover dish with watch 
glass and add ca 15 mL HCl (1 + 1). Let stand overnight, or 
heat on H 2 bath with agitation until ash dissolves. Filter thru 
Whatman No. 30 paper into 125 mL erlenmeyer. Rinse filter 
with enough hot H 2 0, in several portions, to obtain ca 60 mL 
filtrate. 

D. Preparation of Standard Curve 

Dissolve 0.660 g As 2 3 in 25 mL 10% NaOH soln, dil. to 
1 L with H 2 0, and mix. Dil. 10 mL aliquot to 1 L with H 2 
(1 mL = 5 |xg As). Transfer 0, 2, 4, 6, 8, 10, 12, and 14 mL 
aliquots from buret into 125 mL erlenmeyers. Dil. each to ca 
60 mL with H 2 and proceed as in 957. 22E. Plot A against 
jxg As. 

E. Arsine Evolution 

Add ca 10 mL HCl, 2 mL Kl soln, (15%: keep in dark; 
discard when soln turns yellow), and 0.5 mL SnCL soln, (c). 
Swirl, heat in H 2 bath 5 min, and cool. Have all parts of 
evolution app. ready for immediate assembly, with ca 20 mL 
absorbing soln, (d), in 50 mL vol. flask or centrf. tube marked 
at 50 mL. Add 5-6 g Zn, 30 mesh, to digested soln; quickly 
insert stopper contg glass tubing into erlenmeyer and place de- 
livery tube against bottom of vol. flask or centrf. tube so that 
bubbles will be small. Use few drops of H 2 to test for leaks 
between rubber stopper and erlenmeyer. Connecting glass tube 
must be large enough so bubbles will not carry over Pb compds 
from impregnated glass wool plug into absorption flask. 

F. Color Development 

After 30 min, disconnect rubber tubing, leaving delivery tube 
in receiving vessel so that any Hg arsenide on tube will be 
exposed to color-developing reagents. Add 1.0 mL NH 4 
molybdate reagent, (e), and mix by forcing air thru delivery 



AOAC Official Methods of Analysis (1990) 



BUQUINOLATE 



95 



tube. Add 1.0 mL hydrazine sulfate reagent, (f), and again 
mix. Heat in boiling H 2 bath 20 min. Rinse delivery tube 
with H 2 and remove. Cool to room temp., dil. to 50 mL, 
and mix. Filter thru tight glass wool plug in funnel or centrf. 
(Do not use filter paper, as color will be adsorbed.) Read A 
against H 2 at 5:750 nm. Max. A is at 840 nm. Det. As con- 
tent from std curve. 

As x 2.90 = arsanilic acid; As X 2.24 - arsenosobenzene; 
As X 3.51 = 3-nitro-4-hydroxyphenyIarsonic acid; As x 3.3 
= 4-nitrophenyl arson ic acid; As X 3.47 = /?-ureidobenzenear- 
sonic acid. 

Refs.: Ind. Eng. Chem. Anal. Ed. 15, 408(1943); 24, 
1821(1952). Sandell, "Colorimetric Determination of 
Traces of Metals," 3rd ed., 1959. JAOAC 40, 
455(1957). 

CAS-7440-38-2 (arsenic) 



960.62* Bithionol in Feeds 

Spectrophotometric Method 

First Action 1960 

Final Action 1961 

Surplus 1970 



See 38.035-38.037, 11th ed. 



967.34 Buquinolate in Feeds 

Fluorometric Method 

First Action 1967 
Final Action 1972 

A. Principle 

Buquinolate is extd from feed with CHC1 3 , coned to small 
vol., and sepd from interfering substances by TLC utilizing 2 
solv. systems. Buquinolate is eluted from substrate and detd 
fluorometrically. 

B. Reagents 

(a) Alcohol, 80%.— Dil 84.3 mL alcohol to 100 mL with 
H 2 0. 

(b) Developing solvent. — Mix CHC.l 3 with alcohol (10 + 
1). Prep, fresh daily. 

(c) Buquinolate std solns. — (/) Stock soln. — 0.5 mg/mL. 
Dissolve 50.0 mg Buquinolate Ref. Std (available from Nor- 
wich Eaton Pharmaceuticals, Inc., 17 Eaton Ave, Norwich, 
NY 13815) in CHC1 3 to make 100 mL. Warm mixt. on steam 
bath as necessary. Soln is stable 1 month if protected from 
evapn. (2) Working soln. — 100 jmg/mL. Pipet 5 mL stock soln 
into 25 mL vol. flask, dil. to vol. with CHC1 3 , and mix well. 
Prep, fresh daily. 

C. Apparatus 

(a) Developing tanks. — Line developing tanks (for plates 
<20 x 20 cm) with Whatman 3 MM paper. Add 100 mL 
CHC1 3 to one tank; add 100 mL developing solv., (b), to sec- 
ond tank. Prep, each tank fresh daily. 

(b) Plates for TLC. — Clean plates thoroly with alkyl ben- 
zene sulfonate- type detergent (Ajax, or equiv.) and brush; rinse 
plates with H 2 and then with acetone. Let plates air dry. Slurry 
60 g silica gel G (Brinkmann No. 68-00-261-3) with 120 mL 
H 2 0. Pour into suitable applicator and spread 0.500 mm layer 
on 20 x 20 cm plates. Air dry 15-30 min; then dry 2 hr at 
110°. Cool and store plates in desiccator until used. 

(c) Fluorometer. — Either spectrophotofluorometer or filter 
fluorometer may be used. (Suitable filters are: excitation, PTR 



Optics type UV-7E (UV Spectrum Filter, No. 59-07-9, PTR 
Optics Corp., 145 Newton St, Waltham MA 02154); emis- 
sion, Kopp Glass Co. filters C7380 and C5840.) (Caution: See 
safety notes on photo fluoro meters.) 

D. Determination 

(Caution: See safety notes on hazardous radiations.) 

Grind ca 100 g sample to pass No. 30 sieve and mix thoroly. 
Accurately weigh sample contg 1.25 mg buquinolate into 250 
mL g-s erlenmeyer. Pipet 100 mL CHCl 3 into sample flask. 
Shake mech. 1 hr. Filter ext thru Whatman No. 54 paper on 
buchner with mild vac. (Take care to prevent solv. loss by 
evapn.) Transfer exactly 80 mL ext to 150 mL beaker and 
evap. almost to dryness on steam bath. Take up residue in 
small portion CHC1 3 and transfer to 10 mL vol. flask with 
small portions CHC1 3 . Dil. to vol. with CHC1 3 and mix well. 

Apply 250 fxL sample ext and 250 |xL working std soln to 
TLC plate. Place spots ca 25 mm from bottom of plate and 
40 mm apart. (Do not touch pipet to plate.) Develop plate in 
CHCI3 developing tank, (a), until solv. front nearly reaches 
top of plate (ca 1 hr). Observe plate under short wavelength 
UV light: Buquinolate remains at origin; feed background mi- 
grates. Transfer air-dried (5-10 min) plate to tank contg de- 
veloping solv., (b). Let plate develop until solv. front ad- 
vances 12 cm. Air dry 5-10 min. Examine plates under short 
wavelength UV light. Buquinolate migrates from origin (R { , 
0.4-0.6). With spatula, outline each buquinolate spot plus blank 
spot of equiv. area and R { . Remove adsorbent from around 
buquinolate spots and discard. Quant, transfer each spot to sep. 
g-s 25 mL erlenmeyers. Pipet 10 mL 80% alcohol, (a), into 
each flask, shake mech. 20 min, and centrf. 

Det. intensity of fluorescent radiation (7) of sample, std, and 
blank in 10 X 10 mm silica cells, at excitation and emission 
wavelengths of 265 and 375 nm, resp. 



% Buquinolate 

1A * sample 



4iank)/(^sl 



k )j X (0. 1 25 /g sample) 



Ref.: JAOAC 50, 264(1967). 
CAS-5486-03-3 (buquinolate) 



963.33* Cadmium Anthranilate 

in Feeds 
Spectrophotometric Method 

First Action 1963 

Final Action 1964 

Surplus 1974 

See 42.046-42.047, 12th ed. 



977.35 Carbadox in Feeds 

Spectrophotometric Method 
Final Action 1981 

(Applicable to levels >0.0055%. Carbadox solns are light sen- 
sitive. Exts must be protected from direct sunlight or artificial 
light.) 

A. Apparatus 

(a) Filter aid. — Celite 545 (Manville Filtration and Min- 
erals) or Millipore prefilter pad (No. AP2504700, Millipore 
Corp., Ashby Rd, Bedford, MA 01730), or equiv. 

(b) Spectrophotometer. — For use at 520 nm. 



96 



Drugs in Feeds 



AOAC Official Methods of Analysis (1990) 



B. Reagents 

(a) Carbadox std solns. — (J) Stock soln. — 1.10 mg/mL. 
Weigh 110.0 mg Carbadox Ref. Std (available from Pfizer, 
Inc., Quality Control, Agricultural Div., 1 107 S Rt 291, Lee's 
Summit, MO 64048) into 100 mL vol. flask, dissolve in CHC1 3 - 
MeOH (3 + 1), and dil. to vol. with same sol v. Ultrasonic 
bath speeds dissoln. Prep, fresh daily. (2) Working soln. — 
0. 1 10 mg/mL. Pipet 10 mL stock soln into 100 mL vol. flask, 
dil. to vol. with CHCI 3 -MeOH (3 + 1), and mix well. Prep. 
fresh daily. 

(b) Methanolic hydrochloric acid: soln. — IN. Dil. 85 mL 
HCl'to 1 L with MeOH. 

(c) Methanolic sodium hydroxide soln. — 0.057V. Dissolve 
2.0 g NaOH in MeOH and dil. to 1 L with MeOH. Prep, fresh 
weekly or sooner if ppt forms. 

(d) Potassium phosphate soln. — \M. Dissolve 136gKH 2 P0 4 
in H 2 and dil. to 1 L. 

(e) Sodium hydroxide -sodium chloride soln. — Dissolve 100 
g NaCl in 0.17V NaOH and dil. to 1 L with OAN NaOH. 

(f) Stannous chloride soln. — Prep, immediately before use. 
Add 8.0 g SnCl 2 .2H 2 to 100 mL methanolic IN HC1. Place 
in 55-60° H 2 bath and swirl intermittently until soln is clear 
(ca 20 min). Stopper and cool to room temp. Use within 2 hr. 

C. Preparation of Samples 

(Caution: See safety notes on chloroform and methanol.) 

Weigh duplicate portions ground feed into 250 mL erlen- 
meyers: 2.000 g for 0.0330-0.0606% carbadox; 5.000 g, 
0.0110-0.0330%; and 20.00 g, 0.0055-0.0110%. Wet each 
portion with 10 mL H 2 0, let stand 5 min, and add 140 mL 
CHCl 3 -MeOH (3 + 1), Add 15.0 mL working std soln to 1 
portion. Stopper both flasks loosely or with polyethylene stop- 
per with pinhole, and boil gently 1 hr. Cool to room temp. 

Using three 25 mL portions CHCl 3 -MeOH (3 + 1), quant, 
transfer mixt. to buchner precoated with Celite or contg pre- 
f ilter pad, collecting filtrate under vac. in 250 mL vol. flask. 
Dil. to vol. with CHCl 3 -M.eOH (3 + 1), and mix well. Pipet 
100 mL aliquot into 250 mL separator contg 50 mL NaOH- 
NaCl soln. Shake 10 sec and discard lower CHC1 3 layer. Add 
50 mL CHCI 3 , shake 10 sec, and discard CHC1 3 layer. Add 
10 mL KH 2 P0 4 soln, and ext with three 50 mL portions CHC1 3 , 
combining exts in r-b flask. Do not let any solids at interface 
drain into flask. Evap. to dryness, using rotary evaporator and 
60° H,0 bath. 

Conduct reagent blank of H 2 and CHCl r MeOH (3 + 1) 
thru boiling, filtration, extns, and evapn, omitting addn of feed 
and carbadox. 

Alternatively, weigh samples as above and ext as follows: 
Wet each portion with L0 mL H 2 0, let stand 5 min, and add 
140 mL CHCla-MeOH (3 + 1) to one flask. Prep, spiked sam- 
ple by adding 130 mL CHCl 3 -MeOH (3 + 1) and 10 mL car- 
badox working std soln to other flask. Break up any clumps 
with spatula. Stopper tightly and let stand overnight in dark at 
room temp. Using three 25 mL portions of CHCl 3 -MeOH (3 
+ 1) quant, transfer mixt. to buchner precoated with Celite or 
contg pref ilter pad, collecting filtrate under vac. in 250 mL 
vol. flask for unspiked sample and in 500 mL vol. flask for 
spiked sample. Proceed as above, beginning "Dil. to vol. with 
CHCl 3 -MeOH ..." 

D. Determination 

Dissolve residue in flask from sample, sample plus std, and 
blank in 5.00 mL 0.05/V methanolic NaOH. Add 20.0 mL SnCl 2 
soln, swirl gently, and let stand 10 min for complete color 
development. If necessary, clarify soln by filtration thru small 
glass wool plug. If alternative overnight leach was used, clar- 



ify soln by centrifg 10 min at 100 rpm. Within 15 min after 
completion of color development, det. A of clear solns at 520 
nm against MeOH as ref. sol v. Subtract A of blank from A of 
sample and A' of sample plus std, 

% Carbadox = (A/g sample) x [\/(A' -A)] 
X (mg carbadox /mL working std soln) 

x (l g/1000 mg) x 15 mL aliquot X 100 

When alternative overnight leach was used, change calcn to: 

% Carbadox = (A/g sample) x [1/(2 A' - A)] 
x (mg carbadox/mL working std soln) 

X (1 g/1000 mg) X 10 mL aliquot X 100 

Refs.: JAOAC 60, 1059(1977); 62, 982(1979). 
CAS-6804-07-5 (carbadox) 



969.55 Decoquinate in Feeds 

Fluorometric Method 

First Action 1969 
Final Action 1972 

A. Principle 

Decoquinate is extd from feed with 1% CaCL-MeOH soln. 
After addn of H 2 and acid, drug is extd into CHC1 3 , then 
sepd from interfering materials by chromatgy on Florisil. De- 
coquinate is eluted from column with 1% CaCL-MeOH and 
detd by fluorometry against std treated similarly. 

B. Reagents 

(a) Calcium chloride-methanol soln. — 1%. Dissolve 10 g 
anhyd. CaCl 2 , reagent grade, in 1 L MeOH, spectral grade 
(EM Science OmniSolv No. MX0488), or equiv. redistd, re- 
agent grade, anhyd. MeOH. Filter thru Whatman No. 2 paper. 

(b) Decoquinate std solns. — (1) Stock soln. — 300 |mg/mL. 
Weigh 30 mg Decoquinate Ref. Std (available from Hess & 
Clark Inc.). Dissolve and dil. to 100 mL with .1% CaCU-MeOH 
soln. Prep, fresh monthly. (2) Working soln. — 6 [xg/mL. Pi- 
pet 5 mL stock soln into 250 mL vol. flask and dil. to vol. 
with 1% CaCl 2 -MeOH soln. Checks of this soln in 1 cm quartz 
cells at 265 nm against spectral grade MeOH (ca 0.660). Prep, 
fresh std when A is outside range 0.620-0.700. Soln is stable 
>1 week. (3) Fluorescence reference soln. — 1.5 [xg/mL. Pi- 
pet 25 mL working std soln into 100 mL vol. flask and dil. 
to vol. with spectral grade MeOH. Check A at 265 nm as above. 
Prep, fresh std when A is outside range 150-0.190. 

(c) Florisil. — 100-200 mesh (Fisher No. F-101). 

C. Apparatus 

(a) Chromatographic columns. — Draw 30 cm length of 9 
mm tubing (7 mm id) to drip tip. Insert small glass wool plug 
to support adsorbent. Close drip end with short piece of tubing 
and pinch clamp. Add 5 mL CHC1 3 to column, then 0.5 ± 
0.01 g Florisil. Add 2 mL addni CHC1 3 and stir with thin glass 
rod to settle adsorbent. Remove tubing and wash down sides 
of tube with CHC1 3 . Prep, just before use. 

(b) Separators. — 125 or 250 mL with Teflon stopcocks. 

(c) Fluorometer. — (Caution: See safety notes on photo- 
fluorometers.) Either spectrofluorometer or filter fluorometer 
may be used. Excitation filter: UV-2 (UV Spectrum Filters 
No. 14-16-8, 325 nm); emission filter: S/UV (UV Spectrum 
Filters No. 14-01-4, 390 nm) (PTR Optics Corp., 145 Newton 
St, Waltham, MA 02154). 

D. Determination 

Weigh 10 g sample into 125 mL erienmeyer, add exactly 
50 mL 1% CaCL-MeOH soln, stopper, and shake mech. 20 



AOAC Official Methods of Analysis (1990) 



DlBUTYLTIN DlLAURATE 



97 



min. Decant soln into centrf. tube and centrf. 5 min at mod- 
erate speed. Pipet 10 mL clear supernate into 125 mL sepa- 
rator. Prep, std by pipetting 10 mL working std soln into an- 
other separator. Add exactly 10 mL CHC1 3 to each funnel by 
pipet and swirl to mix. Add 100 mL dil. HC1 (1 + 19) to each 
funnel. Shake gently by inverting 25 times; then allow 15 min 
for phases to sep. Drain CHC1 3 layer into centrf. tube and centrf. 
5 min. Remove by aspiration any droplets of floating H 2 phase 
that seps. 

Pipet 5 mL CHC1 3 sample soln onto Florisil column. Pipet 
5 mL CHC1 3 std soln onto another column. Pipet 5 mL CHC1 3 
onto third column (reagent blank). Pass two 10 mL portions 
anhyd. MeOH thru each column. Let MeOH drain to surface 
of Florisil and discard column effluent. Elute with 15 mL 1% 
CaCl 2 -.M.eOH soln, collecting in tube marked at 15 mL. Mix 
well, centrf. if not clear, and transfer to fluorometer cells. Set 
activation wavelength of fluorometer at 325 nm and emission 
wavelength at 390 nm. Set fluorometer sensitivity with fluo- 
rescence ref. std to give convenient scale reading (e.g., 100). 
Det. fluorescence of samples, std, and reagent blank. Subtract 
reagent blank correction, if any, from reading of std and sam- 
ples. 

% Decoquinate in feed - (0.003 x corrected fluorescence 
of sample) /corrected fluorescence of std. 

Ref.: JAOAC51, 1279(1968). 

CAS- 18507-89-6 (decoquinate) 



977.36 Dibutyitin Difaurate in Feeds 

Atomic Absorption Spectrophotometry Method 

First Action 1977 
Final Action 1979 

A. Principle 

DBTD is extd from feed with CHC1 3 , ext is filtered to re- 
move feed particles, and aliquot is coned in presence of MeOH 
until CHCI3 is removed. MeOH soln is dild and filtered to 
remove feed interference, and Sn is detd by AA using air-C 2 H 2 
flame. 

B. Apparatus and Reagents 

(a) Atomic absorption spectrophotometer. — Double beam, 
operated at 286.3 nm with air-C 2 H 2 flame and direct readout 
using 10 mv recorder. Optimize instrument according to man- 
ufacturer's instructions. 

(b) Hot plate. — Regulated to ±3°. 

(c) Mechanical shaker. — Wrist-action type (Burrell Corp., 
or equiv.). 

(d) Tin std solns. — (/) Stock soln. — 500 (xg/mL. Accu- 
rately weigh ca 0.217 g dibutylin bis(2-ethy1hexanoate) (NIST 
SRM No. 1057 or Eastman Kodak No. 10427, % Sn certified) 
into 100 mL vol. flask, and dissolve and dil. to vol. with MeOH. 
(2) Working soln. — 10 jxg/mL. Pipet 2 mL stock soln into 
100 mL vol. flask, add 1.0 mL HC1, and dil. to vol. with 
MeOH. 

C. Preparation of Sample and Extraction 

Grind sample in high-speed blender to pass No. 20 sieve (ca 
3 min), and mix thoroly. Accurately weigh aliquot contg ca 
10 \ig Sn/mL in final soln (see Table 977.36), and transfer 
to 125 mL erlenmeyer. Add 50 mL CHC1 3 , mix, and place 
flask in 55-60° H 2 bath. Let sample reach bath temp.; then 
stopper tightly. Continue heating addnl 30 min, swirling oc- 
casionally. Remove from H 2 bath and shake mech. 20 min. 
Filter thru Whatman No. 4 paper, and collect >30 mL filtrate 
in 50 mL erlenmeyer. 



Table 977.36 Sample Weights for DBTD-Containing Feeds 



%DBTD 



Feed Sample, g 



0.020 (Polystat) 
0.0375 (Tinostat) 
0.0700 (Wormal) 
0.1400 (Wormal) 



13.00 
7.00 
3.75 
2.00 



Pipet 25 mL filtrate into 100 mL graduated beaker, add 2 
boiling chips and 0.25 mL HO, and cone, to ca 10 mL at 
gentle boil on hot plate. Add 20 mL MeOH and cone, sample 
to ca 10 mL again; then repeat with addnl 20 mL and 30 mL 
portions MeOH. (Raise temp, of hot plate to maintain gentle 
boiling as ratio of MeOH to CHCI3 increases.) Remove from 
heat and let cool to room temp. Transfer MeOH soln to 25 
mL vol. flask, washing beaker and funnel with 2-3 five mL 
portions MeOH, dil. to vol. with MeOH, and mix thoroly. 
Filter thru Whatman No. 42 paper and collect filtrate in an- 
other 25 mL vol. flask. 

Prep, blank by dilg 1 mL HC1 to 100 mL with MeOH. 

D. Determination 

(Caution: See safety notes on AAS.) 

Let spectrophtr warm up thoroly and equilibrate by aspir- 
ating MeOH 15 min, using air-C 2 H 2 flame and triple slot burner 
head. Zero spectrophtr by aspirating blank; then aspirate sam- 
ple and std solns, using conditions given in (a). Repeat se- 
quence for each sample. 

% DBTD - A x C X 50 x 5.32 x 10" 6 x (lOO/A') x W 
= (A/A') x (0.266/W) 

where A and A' refer to sample and std, resp.; C — g std/mL; 
and W - g sample. 

Ref.: J AOAC 60, 1054(1977). 

CAS-77-58-7 (dibutyitin dilaurate) 



956.10* Diethylstilbestrol in Feeds 

Spectrophotometric Method 

First Action 
Surplus 1988 



See 42.059-42.062, I4th ed. 



970.85 Dimetridazole in Feeds 

Spectrophotometric Method 

First Action 1970 
Final Action 1988 

A. Principle 

Dimetridazole is extd from feeds with MeOH, sepd from 
interfering substances by two alumina chromatgc steps, and 
detd spectrophtric at its UV wavelength max. Nihydrazone, 
furazolidone, zoalene, 2-chIoro-4-nitrobenzamide, tylosin, and 
large amts procaine (from procaine penicillin) interfere. 

B. Apparatus and Reagents 

(a) Spectrophotometer. — For use in UV. 

(b) Chromatographic tubes. — 13 x 150 mm and 15 x 250 
mm, constricted at bottom to hold glass wool plug and 6 mm 
od delivery tube. 

(c) Aluminum oxide. — Suitable for chromatgy, 961.24B(b). 
To det. suitability of alumina, perform detn on feed that does 
not contain dimetridazole or other imidazole drugs. If feed ap- 



98 



Drugs in Feeds 



AOAC Official Methods of Analysis (1990) 



pears to contain >0.004% dimetridazole, use another batch of 
alumina. 

(d) / ,2 -Dimethyls -nitroimidazole {dimetridazole) std 
solns. — (/) Stock soln. — 0.1 mg/mL. Weigh 100 mg dime- 
tridazole std into 100 mL vol. flask. Dissolve in H 2 by shak- 
ing frequently ca 20 min. Dil. to vol. with H 2 and mix. Pipet 
20 mL into 200 mL vol. flask, dil. to vol.. with H 2 0, and mix. 
(2) Working solns.— Pipet 5, 10, 20, 30, and 40 mL stock 
soln into sep. 100 mL vol. flasks. Add 5.0 mL 3iV HC1 to 
each, immediately dil. to vol. with H 2 0, and mix. Pipet 5 mL 
each soln and 5 mL 0.10/V NaOH into sep. 50 mL erlenmey- 
ers. Stopper and mix. These solns contain 2.5, 5, 10, 15, and 
20 |JLg dimetridazole/mL. 

C. Preparation of Standard Curve 

Proceed as in 970. 85F, using working std solns and blank 
prepd by mixing 5 mL 0.\5N HC1 with 5 mL 0.107V NaOH. 

Read A against blank for recording or manual spectrophtrs. 
Construct std curve by plotting A against jxg dimetridazole/ 
mL. 

D. Preparation of Sample 

Weigh portion finely ground feed contg 0.5-2.0 mg di- 
metridazole (usually 5 g) into 100 mL vol. flask. Add 70-75 
mL MeOH and place in 60° const temp, bath 30 min. Make 
certain that H 2 level covers flask to ca 3 mm below MeOH 
level. Swirl flask 2 or 3 times during first 5 min to heat evenly. 
Cool to room temp., dil. to vol. with MeOH, and mix. Let 
stand 5-10 min to let coarse feed particles settle. 

E. Chromatography 

Place small glass wool plug in bottom of 250 x 15 mm 
chromatgc tube and add 8 cm layer alumina; pack column tightly 
to prevent streaking. (If streaks enter effluent, pos. bias is in- 
troduced.) Decant methanolic ext onto column so that settled 
feed particles are not disturbed. Collect ca 30 mL eluate in 50 
mL vol. flask. Stopper until ready for use. 

(Note: Dimetridazole sublimes at temps >70°; manner of 
sol v. removal is critical.) For feed contg 0.015% dimetrida- 
zole, pipet 15 mL effluent (4 mL if feed contains 0.06%; 3 
mL if feed contains 0.10%) into 125 mL suction or r-b flask 
and evap. under reduced pressure from H 2 aspirator. If 15 
mL is taken, use hot plate (low heat) or H 2 0-bath to reduce 
to 3-4 mL. Shake to prevent bumping. When vol. approaches 
3-4 mL remove flask from heat and remove last 3-4 mL only 
with heat from palm of hand. Continue shaking to prevent 
bumping. Do not attempt to attain complete dryness because 
part of the 2-3 drops of oily residue is dimetridazole. 

Wash down walls of flask, beginning at base of neck, with 
5.0 mL 0.10AT NaOH. Swirl to wash walls. Let stand 5 min 
and add 5.0 mL 0.157V HO. Swirl to mix and wash flask walls. 
Stopper until ready for chromatgy. 

F. Determination 

Prep, second alumina column by inserting small glass wool 
plug into bottom of 150 x 13 mm chromatgc tube, add 4 cm 
layer alumina, and tap gently to pack column lightly. Pour 
entire 10 mL soln onto column and let pass thru by gravity. 
Collect effluent in 50 mL erlenmeyer. Force out liq. adhering 
to column by applying air pressure with rubber bulb. Swirl 
flask to mix. Stopper until ready to read. Pass blank soln of 
5 mL 0.157V HC1 and 5 mL 0.107V NaOH thru sep. 4 cm alu- 
mina column as above. 

(a) Using recording spectrophotometer . — Fill matched pair 
silica cells with reagent blank and with sample soln (always 
use same cell for blank) and scan from 330 to 310 nm. Read 
A at peak and obtain concn of soln in [xg/mL from std curve. 



% Dimetridazole = [((xg/mL from std curve) 

x diln factor x 100]/(g sample X 10 6 ) 

where diln factor = 66.67 for feeds contg 0.015%; 250.0, 
0.06%; and 333.3, 0.1%. 

(b) Using manual spectrophotometer . — Locate peak A of 
sample soln (ca 318 nm), using matched pair silica cells, and 
set wavelength at peak. Read A of sample and blank solns and 
correct sample for blank. Obtain concn of soln in |ag/mL from 
std curve, and calc. % in feed as above. 

Refs.: JAOAC 48, 301(1965); 53, 646(1970). 

CAS-55 1-92-8 (dimetridazole) 



964.29 Ethopabate in Feeds 

Colorimetric Method 

First Action 1964 
Final Action 1967 

A. Principie 

Ethopabate is extd from feed by 50% MeOH at room temp. 
Clear filtrate is acidified with dil. HC1 and extd with CHC1 3 . 
Most interfering substances (amines, p-aminobenzoic acid, 
procaine) are sepd. CHCI3 ext is washed with Na 2 C0 3 soln to 
remove sulfaquinoxaline, acetyl-(/;>-nitrophenyl) sulfanila- 
mide, and chlortetracycline. Ethopabate is converted to free 
amine by controlled acid hydrolysis. Free amine is diazotized 
and coupled; colored complex is extd with w-BuOH and read 
at 550 nm. 

B. Reagents 

(a) Dilute hydrochloric acid.—0.3N. Dil. 25 mL HC1 with 
H 2 to 1 L. 

(b) Sodium carbonate soln. — Dissolve 40 g anhyd. Na 2 C0 3 
in H 2 and dil. to 1 L. 

(c) Coupling reagent (NED). — Dissolve 50 mg iV-(l-naph- 
thyl)ethylenediamine.2HCl in 25 mL H 2 0. Prep, fresh as 
needed. 

(d) Ethopabate std solns. — (J) Stock soln. — 0.400 mg/mL. 
Weigh 40.0 mg Ethopabate Ref. Std (available from Merck & 
Co.) into 100 mL vol. flask, dissolve in MeOH, and dil. to 
vol. (2) Intermediate soln. — 40 fig/mL. Pipet 10 mL stock 
soln into 100 mL vol. flask, dil. to vol. with aq. MeOH (1 + 
1), and mix well. Stored in tightly stoppered flasks, solns are 
stable >1 month. (3) Working soln. — 16.0 (xg/20.0 mL. Pipet 
5 mL intermediate soln into 250 mL vol. flask, dil. to vol. 
with aq. MeOH (1 + 1), and mix well. 

C. Extraction 

Grind feed sample to pass No. 20 sieve and mix thoroly. 
(High-speed blender grinds most feeds to desired fineness in 
ca 3 min.) Accurately weigh sample contg ca 80 jutg ethopabate 
(do not exceed 20 g). Transfer to 250 mL g-s flask. Add 100.0 
mL aq. MeOH (1 + 1) and mag. stirrer bar, stopper tightly, 
and stir 1 hr. (Mech. shaker that provides vigorous agitation 
may be used.) Centrf. , or filter portion of ext thru fast paper. 
Collect only enough filtrate to supply aliquot for test. If nec- 
essary, store exts overnight at room temp, in tightly stoppered 
flasks. 

D. Removal of Interferences 

Pipet 20 mL clear ext into 50 mL centrf. tube. Add 5.0 mL 
dil. HC1 (1 + 9) and 10 mL CHC1 3 , stopper with polyethylene 
stopper, and shake vigorously 3 min on mech. shaker. Centrf., 
and carefully transfer bottom CHC.I3 layer into clean 50 mL 
centrf. tube, using syringe with long needle. Repeat extn with 
two more 10 mL portions CHC1 3 . Add 10 mL Na 2 C0 3 soln to 



AOAC Official Methods of Analysis (1990) 



Furazolidone 



99 



combined CHC1 3 exts, stopper, and shake 3 min. Centrf. , and 
without disturbing interface, draw off most of top aq. layer, 
using syringe, and discard. Repeat washing with another 10 
mL Na 2 C0 3 soln, discarding washing. Add 10 mL H 2 to CHC1 3 
ext, stopper, shake vigorously ca 1 min, and centrf. Draw off 
aq. layer and discard. Repeat with another 10 mL H 2 0. (To 
avoid loss of drug and low results, do not disturb interface on 
CHC1 3 , and complete extn and washings in shortest time pos- 
sible. Prolonged contact with HC1 and Na 2 C0 3 may cause par- 
tial hydrolysis of ethopabate.) 

Pipet 20 mL aq. MeOH (1 + 1) into 50 mL centrf. tube, 
add 5.0 mL dil. HC1 (1 + 9), and proceed as for sample (re- 
agent blank). 

Pipet 20 mL ethopabate working std soln (16.0 jxg) into 50 
mL centrf. tube, add 5.0 mL dil. HO (1 +9), and proceed 
as for sample (std). 

E. Conversion of Ethopabate to Free Amine 

Quant, transfer washed CHC1 3 exts to sep. 100 mL beakers. 
Rinse each centrf. tube with two 3 mL portions aq. MeOH 
(I 4- 1), adding rinsings to beaker. Place beaker on steam bath 
and evap. CHC1 3 to vol. of ca 2 mL. Add 5.0 mL aq. MeOH 
(1 + 1) and swirl beaker to dissolve completely. 

Quant, transfer soln to r-b centrf. tube. Rinse beaker with 
10, 1.0, and 5 mL portions 0.3N HO. Immerse tube in boiling 
H 2 bath so that level of liq. in tube is just below level of 
H 2 bath. Heat 45 min. Remove tube from hot H 2 bath and 
cool to 10-15° in cold H 2 bath. 

F. Development and Measurement of Color 

Remove tubes from cold H 2 bath. Add 1.0 mL freshly prepd 
0.2% NaN0 2 soln to each tube, mix, and let stand 2 min. Add 
1.0 mL 1.0% NH 4 sulfamate soln, mix, and let stand 2 min. 
Add 1.0 mL NED soln, mix, and let stand 10 min. Add 5.0 
g NaCI and 5.00 mL n-BuOH, stopper, and shake vigorously 
until NaCI dissolves. Centrf., carefully transfer portion of clear, 
colored ale. layer to 1 cm cell, and read A at 555 nm against 
/7-BuOH. Correct for reagent blank. 

% Ethopabate in feed 

= 0.008 x (A - A B )/[(A' ~ A B ) x W] 

where A, A B , and A' refer to sample, reagent blank, and std, 
resp., and W = g original sample. 

Refs.: JAOAC 47, 221(1964); 48, 280(1965). 

CAS-59-06-3 (ethopabate) 



985.51 Furazolidone in Feeds and Premixes 

Liquid Chromatographic Method 

First Action 1985 
Final Action 1988 

(Applicable to premixes contg 2-22% furazolidone and to feeds 

contg 0.005-0.05% furazolidone. Note: Furazolidone solns are 

light-sensitive. Protect exts and stds from direct sun and 

artificial light.) 

(Caution: See safety notes on centrifuges, distillation, pipets, 
acetonitrile, acetone, and dimethylformamide.) 

A. Principle 

Unground premix is extd with DMF, and concn of ext is 
adjusted with 5% tetraethyl ammonium bromide (TEAB) to ca 
55 jmg furazolidone/mL for LC. Complete feed is extd with 
H 2 0~acetone on continuous extn app., solv. is evapd, and res- 
idue is dissolved in DMF. 5% TEAB is added to sep. fat. Ext 
is cooled and clarified for LC. 



B. Apparatus 

(a) Liquid chromatograph. — Instrument capable of main- 
taining const pulseless flow of mobile phase at 0.5-1.5 mL/ 
min. Operating conditions: flow rate 1.5 mL/min; loop injec- 
tion vol. 20 julL; detector sensitivity 0.32 AUFS or adjusted to 
produce working std peak response 60-80% full scale; detec- 
tor wavelength 365 nm (settings from 390 to 405 nm may be 
used to improve selectivity for very low level samples if chro- 
matgc column does not adequately resolve interfering peaks). 

(b) Chromatographic column. — Any reverse phase col- 
umn, Cig or C 8 , with particle size <10 |mm that will produce 
single, sharp furazolidone peak with peak skew <\A. Guard 
column may be used. 

(c) Continuous extraction apparatus. — Goldfisch (Lab- 
conco Corp., 8811 Prospect Ave, Kansas City, MO 64132, 
No. 3001), or equiv. 

(d) Extraction thimbles. — Whatman, single thickness, 19 
x 90 mm (Scientific Products,' Inc., No. E6480-4). 

(e) Sample clarification filter . — 13 mm glass fiber pre-filter 
pads (Gelman No. 66073, available from Fisher Scientific Co., 
No. 09-731 A) inserted in 0.5 in. id 5 mL syringe barrel 
(Pharmseal, Scientific Products, No. S9504-5) or any filtration 
device designed for clarification of aq. chromatgc samples. 

C. Reagents 

(a) Extractants . — DMF, reagent grade, for premixes. Ace- 
tone (reagent grade)-H 2 (93 + 7) for complete feeds. 

(b) Diluent. — 5% tetraethylammonium bromide (5% TEAB, 
w/v) (Eastman Kodak Co., No. 1516) in distd, deionized H 2 0. 
Keep in refrigerator. For correct final conens, warm to room 
temp, before pipetting. 

(c) Furazolidone std solns.— (Y) Stock soln. — Ca 1.1 mg/ 
mL. Accurately weigh 0. 1 10 ± 0.005 g furazolidone std (Hess 
and Clark, Inc., 7th & Orange Sts, Ashland, OH 44805) and 
record exact wt to nearest 0.1 mg (WJ. Transfer into 100 mL 
vol. flask, dissolve and dil. to vol. with DMF (sonication aids 
dissolution). Soln is stable if stored in dark. (2) Intermediate 
soln.—Ca 110 |ULg/mL. Dil. 10.0 mL stock soln to 100 mL 
with DMF. Soln is stable if stored in dark. (3) Working std 
soln. — Ca 55 (xg/mL. Mix 10.0 mL intermediate soln with 
equal vol. 5% TEAB. (Mix equal vols. Do not dil. to vol.) 
Let soln cool to room temp. Prep, daily. Diln of std (D s ) = 
2000. 

(d) Mobile phase.— CH 3 CN (LC quality)-2% HOAc in distd, 
deionized H 2 (20 + 80), or as adjusted to give capacity factor 
(k) of ca 2.5 for furazolidone. 

D. Extraction 

(a) Complete feeds. — Det. approx. sample wt to contain ca 
550 |jLg furazolidone by using formula, sample wt, g = 0.555/ 
% guarantee. Accurately weigh (to nearest 0.01 g) calcd wt 
of ground, mixed sample (±5%) into extn thimble (W u = ac- 
tual sample wt). Press cotton plug down onto top of feed to 
prevent channeling. Add 45-50 mL acetone-H 2 (93 + 7) 
extractant and 2 or 3 boiling chips to extn beaker. Ext 8-18 
h on extn app. at "Hi" setting. Evap. solv. on steam bath (stream 
of air directed into beakers or blowing across beakers, as with 
partially closed hood door, hastens evapn). if any H 2 is ev- 
ident in beaker after initial evapn, add ca 25 mL acetone, swirl 
to mix, and re-evaporate (repeat as necessary to remove all 
H 2 0). Remove from steam bath as soon as evapn is complete. 
Add 5.00 mL DMF, heat on steam bath just until bottom of 
beaker is hot (15-30 s) and swirl, washing sides, to dissolve 
residue (generally all residue dissolves in warm DMF). Add 
5.00 mL 5% TEAB, mix, pour soln into 15 mL centrifuge 
tube, and let cool. Centrifuge 5-10 min at 2000 rpm (2500 X 
g). Using disposable pipet attached to aspirator and trap, re- 



100 



Drugs in Feeds 



AOAC Official Methods of Analysis (1990) 



Table 985.51 Sample Sizes, Dilutions, and Total Sample 
Dilutions for Assay of Furazolidone in Feeds 









Total 


Label 






Sample 


Claim, 


Sample Wt, 


Dilns with 


Diln (D u ), 


% 


g 


DMF, mL 


mL 


2.2 


1.00 


none 


400 


3.3 


1.00 


30/50 


666.7 


11.0 


1.00 


20/100 


2000 


22.0 


1.00 


10/100 


4000 



move fat layer floating on supernate. Diln for complete feed 
samples (£> u ) = 10. 

(b) P remixes. — Accurately weigh (±5%) amt unground 
sample indicated in Table 985.51 into 500 mL g-s erlenmeyer 
(W u = actual sample wt). Add by pipet 200.0 mL DMF, stop- 
per, and shake flask 30 min. Either let suspended material set- 
tle or centrf. or filter portion of ext. Dil. with DMF to ca 1 10 
jjig furazolidone/mL. To 5.00 mL 5% TEAB soJn add 5.00 
mL dild ext, mix, and let soln cool to room temp. Clarify as 
for complete feeds, (a). (Total sample diln = D u , Table 985.51.) 

E Determination 

(a) Complete feeds, — Make several injections of furazoli- 
done working std soln, adjusting mobile phase strength to give 
k ca 2.5 and peak ht 60-80% full scale (k = (A, - t )/t , where 
t = distance from injection to first perturbation of std chro- 
matogram). Make 2 or more injections of std to ensure 1-2% 
repeatability of peak responses. Bracket each 2 sample injec- 
tions by std injections. Use av. peak ht P s (or av. peak area) 
of stds bracketing each pair of samples to calc. furazolidone 
concn in samples (sample peak response = P u ). If no drift in 
std peak hts is evident thruout run, then use av. for all std 
injections in calcns. 

(b) P remixes. — Det. furazolidone as for complete feeds (a). 
Diln (£> u ) is shown in Table 985.51. 

F. Caicuiation 

% Furazolidone 

= (Pu x W s x D u x ]00)/(P s x W u x D s ) 

where P u and P s = peak response of sample (unknow r n) and 
std, resp.; W u and W s - g sample and std, resp,; and D u and 
D & - mL total dilns of sample and stds, resp. Det. total dilns 
as in following example: If extn of 1 g sample in 200 mL solv. 
is followed by serial dilns of 20/100 and 5/10, then total diln 
is 1 g/200 mL X 20/100 X 5/10 - 1 g/2000 mL, and D u 
= 2000 mL. 

Ref.: JAOAC68, 1033(1985). 

CAS-67-45-8 (furazolidone) 



960.63 Furazolidone, Nitrofurazone, 

or Bifuran in Feeds 
Colorimetric Method 

First Action 1960 
Final Action 1961 

A. Reagents 

(a) P he ny [hydrazine hydrochloride soln. — Dissolve 0.5 g 
phenylhydrazine.HCl in 50 mL H 2 0. Prep, fresh daily. Mix 
equal vol. of this soln with HC1. 

(b) Furazolidone std solns . — (I ) Stock soln . — . 55 mg/mL . 
Weigh 55 mg furazolidone std (available from Hess & Clark, 
Inc.) into 100 mL vol. flask, dil. to vol. with dimethylform- 



amide (DMF), and mix. Soln is stable several months when 
protected from light. (2) Working soln. — Prep, working std 
corresponding to label declaration. For feeds contg 0.011% 
furazolidone, pipet 2 mL stock soln into 100 mL vol. flask, 
add 48 mL DMF, and dil. to vol. with H 2 0. For feeds contg 
0.00275% furazolidone, pipet 0.5 mL stock soln into 100 mL 
vol. flask, add 49.5 mL DMF, and dil. to vol. with FLO. 

(c) Nitrofurazone std solns.— (J) Stock soln. — 0.56 mg/mL. 
Weigh 56 mg nitrofurazone std (available from Hess & Clark, 
Inc.) into 100 mL vol. flask, dil. to vol. with DMF, and mix. 
Soln is stable several months when protected from light. (2) 
Working soln. — Prep, working std corresponding to label dec- 
laration. For feeds contg 0.0056% nitrofurazone, pipet 1 mL 
stock soln into 100 mL vol. flask, add 49 mL DMF, and dil. 
to vol. with H 2 0. For feeds contg 0.01 12% nitrofurazone, pi- 
pet 2 mL stock soln into 100 mL vol. flask, add 48 mL DMF, 
and dil. to vol. with H 2 0. 

(d) Bifuran std solns. — (J) Stock soln. — 0.1285 mg/mL. 
Pipet 20 mL nitrofurazone stock soln and 3 mL furazolidone 
stock soln into 100 mL vol. flask and dil. to vol. with DMF. 
(2) Working soln. — For feeds contg 0.0064% total nitrofur- 
ans, prep, working std by pipetting 5 mL bifuran stock soln 
into 100 mL vol. flask, adding 45 mL DMF, and dilg to vol. 
with H 2 0. 

(e) Adsorbent. — To 100 parts alumina, 961.24B(b), in screw 
cap bottle, add 4 parts Mg(OH) 2 , shake until thoroly mixed, 
then add 5 parts H 2 0, and mix until all lumps disappear. Store 
in tightly sealed container. 

B. Determination 

(Caution: See safety notes on pipets, toxic solvents, and di- 
methylformamide.) 

Grind coarse or pelleted feeds to "20 mesh" thru cutting- 
type mill such as Wiley Intermediate. Finer feeds need not be 
ground. Weigh 10 g sample into 125 mL erlenmeyer, add ex- 
actly 50 mL DMF, stopper loosely, and place in boiling H 2 
bath 5 min. Mech. shake 10 min and filter thru rapid paper. 
To 25 mL filtrate add 25 mL H 2 and mix. 

Prep, ca 20 mm diam. adsorption column, contg adsorbent, 
to ht of 5 cm. Pass the 50% DMF sample soln thru column, 
discarding first 3 mL eluate. (If column flow stops, break up 
gummy film at top of adsorbent, using Jong thin glass rod.) 
Pipet 5 mL aliquots of eluate into each of 2 numbered test 
tubes. Protect one tube from light. To other tube, add 3 drops 
freshly prepd 2% soln ofNa hydrosulfite and let stand 20 min, 
shaking at ca 5 min intervals. Treat 5 mL aliquots of working 
std soln in exactly same manner. 

Pipet 5 mL phenylhydrazine.HCl soln into each of the num- 
bered test tubes contg samples and stds. Mix and place tubes 
in 70° H 2 bath 25 min; cool in 15° H 2 bath 5 min. Add 
exactly 10 mL toluene to each tube, stopper, and shake vig- 
orously 40 times. Centrf. or filter toluene soln directly into 
absorption cell thru cotton wad inserted in stem of small fun- 
nel. Read A of solns at 440 nm. 



% Furazolidone 

Lv*samp. ^"* red. samp. 

% Total nitrofurans (bifuran) 



.) x 0.011 (or0.00275)]/(A nd - A icdslc] ) 



I, v* samp. ^ red. samp 

% Nitrofurazone 

t\/*samp. ^red.samp. 



) x 0.0064]/(/\ s 



) X 0.0056 (or 0.011)]/(A std - A rcd , td ) 



Refs.: JAOAC 40, 463(1957); 41, 333(1958); 43, 310(1960); 
44, 30(1961); 52, 233(1969). 

CAS-67-45-8 (furazolidone) 
CAS-59-87-0 (nitrofurazone) 



AOAC Official Methods of Analysis (1990) 



Ipronidazole 



101 



973.80 



Furazolidone 
and ZoaBene in Feeds 

Qualitative Tests 

First Action 1973 
Final Action 1988 



A. Apparatus 
See 964.07A. 



B. Reagents 

(a) Dimethylformami.de {DMF). — Reagent grade. 

(b) Alcoholic potassium hydroxide soln. — 4%. Dissolve 4 
g KOH in 100 mL alcohol. If premixed with DMF (1 + 9), 
prep, fresh daily. 

(c) Ethylene diamine . — Use in hood. 

C. Preparation of Sample 

Gently grind pellet, cube, or crumble forms with mortar and 
pestle. Sieve thru nest of Nos. 10, 20, and 30 sieves with pan. 
Drugs usually are coned in portion in pan. 

D. Identification 

(a) DMF test. — Place 9 drops DMF and 1 drop ale. KOH 
soln in each of 3 depressions of white spot plate. Sprinkle ca 
0.01 g fine feed material into each soln from tip of spatula 
while observing reaction under microscope. Furazolidone pro- 
duces intense blue color, easily detected at > 0.0025%. Zo- 
alene gives bright green, easily detected at >0.0025%. Color 
of minute particles of zoalene fades rapidly; color of larger 
particles persists 3-5 min. 

(b) Ethylenedi amine test. — Place dry filter paper at bottom 
of petri dish and sprinkle ca 0.5 g fine feed evenly over paper. 
Dispense 2-4 mL ethylenediamine under edge of paper so as 
to wet entire paper and sample. Examine under stereoscopic 
microscope at 10x for particles developing bright purple, in- 
dicating zoalene, or deep red, indicating furazolidone. (Blood 
meal, frequently used in livestock feed, also gives deep red 
color with reagent.) 

Refs.: JAOAC 56, 762(1973); 60, 389(1970). 

CAS-67-45-8 (furazolidone) 
CAS- 148-0 1-6 (zoalene) 



960.64* Glycarbylamide in Feeds 

Colorimetric Method 

First Action 1960 

Final Action 1961 

Surplus 1970 

&?<? 38.066-38.071 , 11th ed. 

971.44 Ipronidazole in Feeds 

Gas Chromatographic Method 
First Action 1971 

A. Principle 

Ipronidazole is extd from feed with warm 0.2JV HCl, trans- 
ferred to benzene after alkalinization, and measured by GC 
with electron capture detector. 

B. Apparatus 

(a) Gas chromatography — With electron capture detector. 
Conditions: temps (°) — column 180 ± 2 (isothermal), injec- 
tion 240 ± 2, detector 210 ± 2; N How 60 mL/min (install 
3' (0.9 m) x V/ molecular sieve 5 A trap in N line). 

(b) Recorder. — 0- I mv input and l.O sec full scale de- 
flection, chart speed 0.75"/min, or equiv. 



(c) Gas chromatographic column. — 0.9 m (3') X l /s" od 
stainless steel tubing packed with 5% Carbowax 20M-tere- 
phthalic acid (TPA) on 100™ 120 mesh Chromosorb G, acid- 
washed and dichlorodimethylsilane-treated (Applied Science). 

(d) Column preparation. — Dissolve 1.0 g Carbowax 20M- 
TPA in 30 mL CHC1 3 and let stand over 19.0 g Chromosorb 
G 3 days with occasional swirling. After standing, evap. CHC1 3 
under N and dry 1 hr in 70° oven. After packing, condition 
column 2 days at 200° in slow stream of N before use. 

C. Reagents 

(a) Benzene. — Redistil from all-glass app., discarding first 
and last 10%. (Caution: See safety notes on distillation, flam- 
mable solvents, toxic solvents, and benzene.) 

(b) Ipronidazole. — Available from Hoffmann-La Roche, 
Inc.; prep, std soln contg 0.3 fxg ipronidazole/mL benzene. 

D. Chromatography of Standard 

Inject 5 )ulL std ipronidazole soln into gas chromatograph 
and adjust conditions to obtain ca 5 min retention time. Det. 
area under ipronidazole peak as product of peak ht (cm) and 
width (cm) at half ht, using slope baseline technic. After sat- 
isfactory performance of column is established, inject at least 
duplicate 5 |xL aliquots of std soln (1.5 ng/5 |xL) at beginning 
of work day and periodically thereafter (e.g., after each 5-6 
sample injections). Measure ipronidazole peak area in each case. 

E. Preparation of Sample 

(Caution: See safety notes on pi pets.) 

Ext mash-type feeds without prior grinding. Grind pelleted 
feeds to pass No. 30 sieve before extn. (Method for feeds is 
described for 0.0060% level of ipronidazole with modifica- 
tions indicated for 0.0030 and 0.0090% levels.) 

Transfer 10 g sample into 500 mL vol. flask (for 0.0030% 
level, use 20 g sample) and add exactly 200 mL 0.27V HCl 
previously warmed to 40 ± 3°. Stopper flask and shake mech. 
20 min. Let settle few min. Centrf. portion of supernate and 
pipet 10 mL (for 0.0090% level, transfer 5.0 mL and add 5 
mL 0.2/V HCl) to g-s centrf. tube. Place tube in cold H 2 bath 
and add 0.5 mL 5N NaOH to make alk. (pH - 10-12.5; check 
with Accutint pH paper; do not hold el u ate at high pH for 
prolonged time). Pipet 20 mL benzene into tube, stopper, shake 
mech. 5 min, and centrf. 10 min at 2500 rpm. Pipet 5 mL 
clear benzene ext into 25 mL vol. flask, dil. to vol. with ben- 
zene, and mix. This is sample soln. 

F. Determination 

Inject 5 uX sample soln into chromatograph. Measure area 
of ipronidazole peak obtained from chromatogram. 

% Ipronidazole = (B x C x D)/(B' x 10,000) 

where B and B' = areas under ipronidazole peak for sample 
and std solns, resp.; C = p,g ipronidazole/mL in std soln (0.3); 
and D = diln factor (0.0030% level, D - 100; 0.0060%, 200; 
and 0.0090%, 400). 

Ref.: JAOAC 54, 72(1971); 57, 29(1974). 

CAS- 14885-29- 1 (ipronidazole) 



980.36 Melengestrol Acetate 

in Feed Supplements 
Gas Chromatographic Method 

First Action 1980 
Final Action 1988 

(Caution: See safety notes on chloroform, hexane, and 
methanol.) 



102 



Drugs in Feeds 



AOAC Official Methods of Analysis (1990) 



A. Principle 

MGA is extd from aq. slurry of supplement sample with 
hexane, partitioned from hexane into aq. MeOH, and then from 
aq. MeOH into CH 2 C1 2 . After evapn of CH 2 CI 2 , dried ext is 
transferred to alumina column with hexane, and eluted with 
CHC1 3 -hexane soln. MGA in el u ate is detd by GC. MGA can 
be assayed in cattle feed supplements contg 0.125 mg/lb to 
1.00 mg MGA/lb supplement (0.28-2.2 mg/kg). 

B. Apparatus 

(a) Extractor. — Liq.-liq. (Ace Glass No. 6840-96 or equiv.). 

(b) Gas chromato graph.— Tracor Model MT-220 (replace- 
ment Model 540, Tracor Instruments Inc.), or equiv., with 
63 Ni pulsed electron affinity detector, 0.6 m (2') x 3 mm id 
glass column packed with 1% OV-17 on 100-200 mesh Gas- 
Chrom Q (Applied Science), and Tracor 1.0 mv recorder with 
chart speed of 0.5"/m.in. Operating conditions. — Linde 
99.996% high purity N, or equiv., carrier gas 50 mL/min; 
purge, off; temps (°) — injector 235, detector 300, column 225; 
detector pulse ht 60 V, pulse interval 300 jxsec, sensitivity 80 
x 10"" AFS. 

(c) Rotary-evaporator. — Valley Electromagnetics, One 
Wolfer Park, Spring Valley, IL 61362, or equiv., 976.36B(m). 

(d) Chromatographic tube. — 18 x 500 mm, fitted with 
coarse porosity fritted glass disc and Teflon stopcock (Fischer 
and Porter Co., or equiv.). 

C. Reagents 

(a) Aluminum oxide. — Woelm acid, anionotropic, activity 
grade 1 for column chromatography (ICN Pharmaceuticals or 
equiv.). 

(b) Cholesteryl chloroacetate (CCA) internal std soln. — (I) 
Stock soln. — 500 jig/mL. Dissolve 125 mg (Aldrich Chemi- 
cal Co., C7680-9) in 250 mL absolute ethanol-hexane (5 + 
95). (2) Working soln I. — 50 fig/mL. Pipet 10 mL stock soln 
and dil. to 100 mL with absolute ethanol-hexane. (3) Working 
soln II. — 5 (xg/mL. Pipet 10 mL stock soln and dil. to 1 L 
with absolute ethanol-hexane (5 + 95). 

(c) Medroxyprogesterone acetate {MAP) extraction std 
soln. — (/) Stock soln. — 2 mg/mL. Dissolve 200 mg (The Up- 
john Co., 7000 Portage Rd, Kalamazoo, Ml 49001) in 100 
mL absolute alcohol (requires overnight shaking). (2) Working 
solns. — ie, 20, 30 and 60 |xg/mL. Pipet 8, 10, 15 and 30 
mL aliquots stock soln and dil. to I L with absolute alcohol 
for use with 0.125-0.250, 0.250-0.450, 0.450-0.750, 0.750- 
1.00 mg MGA/lb samples, resp. 

(d) Melengestrol acetate (MGA). — (I) Stock soln. — 25 |jLg/ 
mL. Dissolve 100 mg Ref. Std (The Upjohn Co.) in 100 mL 
absolute alcohol. Dil. 5.0 mL to 200 mL with hexane. (2) 
Working soln. — 1.25 jxg/mL. Dil. 10 mL stock soln to 200 
mL with absolute alcohol-hexane (5 + 95). 

(e) Gas chromatography reference soln. — CCA, 5 |xg/mL; 
MAP 2.4 fxg/mL; MGA, 0.125 u.g/mL. Pipet 10 mL each 
CCA (50 u.g/mL) and MGA (1.25 fig/mL) and 4 mL MAP 
(60 |mg/mL) and dil. to 100 mL with hexane. 

(f) Solvents. — Distd-in -glass hexane and CH 2 C1 2 , pesticide 
and gas chromatgy grade (Burdick & Jackson Laboratories, or 
equiv.). 

(g) Solvent partition soln. — Mix 0.25% aq. Na?S0 4 soln 
with MeOH (30 + 70). 

D. Extraction 

Grind dry samples to pass 1 mm screen. Thoroly mix sample 
and place ca 15 g sample, weighed to nearest 0.01 g, into 
extractor, and pipet 10 mL appropriate extn std soln, (c), for 
level MGA being assayed. Rinse sample to bottom of extractor 
with 30 mL H 2 0. Place mag. stirring bar in extractor. Fill to 



side arm with hexane and insert solv. return tube in position. 
Attach extractor to condenser and 500 mL receiving flask contg 
100 mL hexane and several SiC boiling chips. Ext sample 3 
hr by heating receiving flask enough to have rapid reflux (^20 
mL/min) at condenser while stirring sample vigorously, ft is 
imperative that reflux rate be fast enough to completely ext 
sample in 3 hr. 250 watt Glas-Col heating mantle operated at 
120 v will do this. Control emulsions by regulating stirring 
rate. Sample must be stirred vigorously and continuously. Up 
to 30 mL addnl H 2 may be added thru condenser to aid stir- 
ring action, if necessary. Let apparatus cool, set receiving flask 
aside, and transfer extractor contents to 1 or 2 L separator. Let 
phases sep. clearly; discard aq. (lower) layer and transfer hex- 
ane layer to 2 L r-b flask and roto-evap. just to dryness. Dis- 
solve residue in 100 mL solv. partition soln, (g). 

E. Solvent Partition 

Quant, transfer ext in 500 mL receiving flask to 500 mL 
separator. Rinse 2 L flask with 100 mL solv. partition soln, 
(g), into receiving flask and then quant, transfer receiving flask 
contents into same separator. Gently shake funnel by inverting 
15 times, let solv. layers sep. clearly, and drain lower layer 
into 1 L separator contg 200 mL CH 2 C1 2 . Repeat rinsing flasks, 
transfers, and partitioning with 3 or more 100 mL portions 
solv. partition soln. Vigorously shake combined exts in 1 L 
separator 15 sec, let phases sep, clearly, and drain lower 
(CH 2 C1 2 ) layer into original 500 mL receiving flask. Repeat 
partition into CH 2 CI 2 2 more times, using 40 mL CH 2 C1 2 each 
time. Roto-evap. combined exts to near dryness. Remove re- 
sidual H 2 with >2 sep. addns of 10 mL absolute alcohol and 
roto-evap. solv. Remove residual alcohol with two 10 mL por- 
tions hexane, roto-evapg each portion. Dissolve residue in 10 
mL hexane. 

F. Alumina Column Chromatography 

Prep, column by slurrying 20 g aluminum oxide in CHC1 3 
and transfer with CHC1 3 wash bottle to tube, (d), contg ca 100 
mL CHC1 3 . While column drains, let alumina settle and add 
5 g Na 2 S0 4 to column while letting CHC1 3 slowly drain. Use 
ca 100-150 mL total CHC1 3 in this step. In this and following 
steps, it is important to let each portion of solv. added drain 
to top qfNa 2 S0 4 layer before adding next portion. CHC1 3 must 
contain 0.5-1.0% alcohol. Rinse column with 10-15 mL hex- 
ane, followed by three 50 mL hexane washes. While column 
drains, quant, transfer ext to column with four 10 mL portions 
and one 75 mL portion hexane. Discard all washings collected 
to this point. Elute MGA with five 50 mL portions CHCI3- 
hexane (33 + 67) solv., using each portion to rinse flask contg 
ext; collect 250 mL eluate in 250 mL vol. flask. 

G. Determination 

Thoroly mix column eluate and pipet aliquot for analysis as 
follows: 0.125-0.250 mg MGA/lb, 40 mL; 0.250-0.450 mg 
MGA/lb, 30 mL; 0.450-0.750 mg MGA/lb, 20 mL; 0.750- 
1.00 mg MGA/lb, 10 mL, and evap. to dryness in 50 mL g- 
s r-b flask, using warm (45-60°) hot plate or H 2 bath and 
stream of N or air. When aliquot approaches dryness, add ca 
5 mL absolute alcohol and evap. to remove residual H 2 0. Re- 
peat addn of alcohol if necessary until aliquot dries com- 
pletely. Dissolve in 10.0 mL 5 juig/mL CCA working soln II, 
(b)(3). Inject 3 julL each of sample and std into gas chromato- 
graph operated as in 980.36B(b). Order of elution, min: MAP, 
4-6; MGA, 6-8: CCA, 8-1 1 . Make several injections to achieve 
reproducible (< ± 5%) peak hts or areas depending on type 
and condition of gas chromatograph. // is imperative that GC 
response be linear. Check linearity using MGA cones 0.5 and 
2.0 times GC ref. soln, (e), concn (0.0625, 0.125, and 0.25 
(mg/mL). Keep CCA concn const. 



AOAC Official Methods of Analysis (1990) 



NlCARBAZIN 



103 



H. Calculations 

Calc. mg MGA/lb sample: 

mg MGA/lb 

= R X R' X 1.25 X (250/mL aliquot) X (0.4536/W) 

where R and R' — peak ht ratios of MG A/CCA in sample and 
CCA/MGA in std, resp.; 1.25 - |mg MGA in 10 ml std soln 
(e); 250 = mL eluate collected; mL aliquot = 10-40 mL ali- 
quot taken; 0.4536 - conversion factor to obtain mg/lb; W = 
g sample extd. MAP is added to sample prior to extn to in- 
dicate magnitude of extn efficiency and cleanup losses. Reas- 
say samples showing <85% MAP recovery after reasons for 
low MAP recovery have been detd. 

% MAP recovered 

= R { X R 2 X 24 x (100/Af) X (250/mL aliquot) 

where /? t and R 2 = peak ht ratios of MAP/CCA in sample 
and CCA/MAP in std, resp.; 24 - fxg MAP in 10 mL std soln 
(e); M. = [Jig MAP added to sample; and (250/mL aliquot) are 
defined above. 

Ref.: J AOAC 63, 425(1980). 

CAS-29 19-66-6 (melengestrol acetate) 



971.45* Nequinate in Feeds 

Spectrophotofluorometric Method 

First Action 1971 

Final Action 1974 

Surplus 1975 

See 42.084-42.087, 1 3th ed. 

956.11 Nicarbazin in Feeds 

Spectrophotometric Method 
Final Action 

(Presence of furazolidone, nitrofurazone, or nihydrazone may 

cause high results. Confirm presence of nicarbazin by 

Identification Test, 956. 11F.) 

A. Reagents 

(a) Dimethylformamide (DMF). — Reagent grade. 

(b) Alumina. —See 961.24B(b). 

(c) Alcohol. — Formulas SDA Nos. 2B, 3A, or 30 may be 
used. 

(d) Alcoholic sodium hydroxide soln. — Dil. 2.0 mL clear 
50% NaOH soln, 936.16B(b), to 100 mL with alcohol. Centrf. 
in stoppered tube. Prep, fresh daily. 

(e) Nicarbazin std solns. — (7) Stock soln. — Weigh 25.0 mg 
Nicarbazin Ref. Std (available from Merck & Co.) into 500 
mL vol. flask, and dissolve in ca 150 mL DMF with aid of 
gentle heat. Cool, dil. to vol. with DMF, and mix well. Store 
protected from light. (2) Working soln. — 12.5 |ULg/mL. Trans- 
fer 25.0 mL stock soln to 100 mL vol. flask and dil. to vol. 
with DMF. Mix well. 

B. Preparation of Column 

Use glass tube 22 mm id, ca 50 cm long, constricted at 
lower end. Place plug of glass wool in constricted end and add 
30 g alumina in 3 portions. Tamp each portion with glass rod 
while applying gentle suction. Wash column with 25 mL DMF, 
draining to point 1-2 cm above bed level before adding sample 
to column. Prep, column for each sample and std. 

Never let column run dry; keep head of liq. at all times. 



C. Preparation of Sample 

(Caution: See safety notes on distillation, toxic solvents, and 
dimethylformamide.) 

Weigh 10.0 g sample into 250 mL erlenmeyer and add 100.0 
mL DMF. Heat just to bp on hot plate in hood with intermit- 
tent stirring. Cool to room temp, by immersing in H 2 bath. 
Decant supernate into centrf. tubes and centrf. 3 min. 

D. Determination 

(Caution: See safety notes on pipets.) 

Pipet 25.0 mL clear ext onto column and let pass thru col- 
umn with aid of gentle suction. Wash column with three 10 
mL portions DMF and reject washings. Elute with nine 5 mL 
portions alcohol, discarding first 15 mL eluate and collecting 
next 25 mL eluate in 25 x 200 mm tube. Quant, transfer eluate 
into 50 mL vol. flask and dil. to vol. with alcohol. Mix well. 

Pipet 25.0 mL working std soln onto another column and 
proceed as for sample. 

Pipet two 15.0 mL portions sample soln into sep. 25 mL 
vol. flasks. To one add 5.0 mL ale. NaOH soln and adjust 
vol. of both solns to 25 mL with alcohol. Read A of yellow 
soln formed in first flask within 5 min in spectrophtr or col- 
orimeter at 430 nm against second soln as blank. Calc. wt 
nicarbazin from std curve. 

E. Preparation of Standard Curve 

Pipet 10, 15, and 20 mL aliquots of chromatgd working std 
soln into sep. 25 mL vol. flasks, add 5 mL ale. NaOH, and 
dil. to vol. with alcohol. Mix well. Measure A within 5 min 
at 430 nm against alcohol. 

Prep, std curve by plotting A against jxg nicarbazin. 

F. Identification Test 

Place alcohol in 1 cm quartz cell and clear chromatgd sam- 
ple soln in matched cell. Det. A at 2 nm intervals from 340 
to 349 nm with Beckman Model DU spectrophtr, or equiv., 
at min. slit width. Absorption max. at 344 ± 4 nm confirms 
presence of nicarbazin. 

Refs.: JAOAC 39, 321(1956); 40, 469(1957); 41, 326(1958). 

CAS-330-95-0 (nicarbazin) 



967.35 Nicotine in Feeds 

Spectrophotometric Method 

First Action 1965 
Final Action 1967 

(Applicable in the presence of phenothiazine, dibutyltin di- 
laurate, and 2,2'-dihydroxy-5,5'-dichlorodiphenylmethane) 

A. Principle 

Nicotine is extd with alkali, steam distd, extd with CHCI 3 , 
and detd spectrophtric in acidic soln. 

B. Reagents 

(a) Dilute hydrochloric acid. — 0.05JV. Dil. 4.1 mL HCI to 
1 L with H 2 0. 

(b) Nicotine std soln. — 0.012 mg/mL. Accurately weigh 
ca 100 mg nicotine and dil. to 100 mL in vol. flask with 0.05/V 
HCI. Transfer 3.0 mL aliquot to 250 mL vol. flask and dil. 
to vol. with 0.05N HCI. (Caution: Nicotine is very toxic.) 

(c) Antifoam. — Antifoam A (Dow Corning Corp.), or equiv. 



104 



Drugs in Feeds 



AOAC Official Methods of Analysis (1990) 



C. Apparatus 

(a) Distillation flask. — 250 mL r-b flask and CJaisen distg 
head or 250 mL Claisen flask. 

(b) Condenser. — Graham coil type with 300 mm jacket 
(Corning No. 2500, or equiv.). Must be used in vertical po- 
sition. 

(c) Ultraviolet recording spectrophotometer. — Double beam, 
capable of scanning UV spectrum from 220 to 300 nm, with 
1 cm cells. 

D. Determination 

(Detn can be interrupted at any step where soln is acidic.) 

Accurately weigh representative portion of feed, ground thru 
No. 20 sieve, contg ca 3 mg nicotine and transfer to 250 mL 
centrf. bottle. Add 100 mL 0.5% NaOH soln, stopper (Neo- 
prene or rubber), and shake vigorously 1 min. Centrf. ca 5 
min at 1500 rpm. Decant free flowing and viscous liq. into 
400-600 mL beaker. Rinse lip and centrf. bottle into beaker 
with few mL H 2 0, being careful not to dislodge solid material. 
Repeat extn, centrf g, and rinsing twice, combining supernates 
in beaker. Adjust soln to pH 2-3 with HO and evap. on hot 
plate to ca 100 mL. Cool, adjust to pH 10-14 with NaOH (1 
+ 1), and transfer quant, to distn flask, using min. of H 2 0. 
Vol. must be <125 mL. Add 10 drops antifoam to flask. Place 
tip of condenser below surface of 7 mL H 2 S0 4 (1 +5) in 500 
mL flask or beaker (container must be tilted at first to obtain 
sufficient depth). Steam distil at rate of >:8 mL/min. (It will 
be necessary to heat Claisen flask as distn proceeds to avoid 
condensation of steam in flask.) Collect ca 300 mL distillate. 
When distn is complete, rinse condenser into receiver with ca 
5 mL H 2 0. 

Transfer distillate to 500 mL separator, rinse with H 2 0, make 
distinctly alk. (pH 10-14) with NaOH (1 + 1), and ext with 
five 20 mL portions CHC1 3 . Combine CHC1 3 exts in 250 mL 
separator. Ext nicotine from CHC1 3 with 20, 20, 15, 15, and 
15 mL portions 0.05W HC1. Combine HC1 exts in 125 mL 
separator and shake gently few sec with 15 mL pet ether to 
remove any remaining CHC1 3 . Drain clear HO layer into 250 
mL vol. flask and wash pet ether with addnl 10 mL 0.05./V 
HO. Drain acid layer into vol. flask, dil. to vol. with 0.05W 
HO, and record UV spectrum from 220 to 300 nm in 1 cm 
cell against 0.05/V HO in recording spectrophtr. Draw line 
tangent to 2 min. obtained (ca 226 and 280 nm), drop per- 
pendicular from point of max. A (ca 259 nm) to tangent line, 
and det. net A. Similarly det. net A' of std soln. 

% Nicotine in feed 

= (Net A x mg nicotine in final std soln 

x 100)/(Net A' x g sample x 1000) 

ReL: JAOAC 47, 226(1964). 

CAS-54-11-5 (nicotine) 



971.46 Nifursol in Feeds 

Spectrophotometry Method 
First Action 1971 
Final Action 1984 

A. Principle 

Nifursol is extd from feed with dimethyl formamide (DMF). 
Feed interferences are removed from DMF soln by column 
chromatgy on alumina. Drug is reacted with phenylhydrazine 
to form 5 -nitro furfural phenylhydrazone, which is extd into 
toluene and detd spectrophtric at 555 nm immediately after 
addn of methylbenzethonium hydroxide. 



B. Apparatus 

(a) Chromatographic tubes. — 400 X 20 mm id, with glass 
wool plug. 

(b) Spectrophotometer . — For use at 555 nm. 

C. Reagents 

(a) N ,N-Dimethy Iformamide (DMF) solns. — (1) 95% 
DMF.— Dil. 95 parts DMF (No. DX1730, EM Science or 
equiv.) with 5 parts H 2 0. (2) 50% DMF.— Dil. 50 parts DMF 
with 50 parts H 2 0. (Caution: See safety notes on dimethyl- 
formamide.) 

(b) Alumina. — To 100 parts 80-200 mesh alumina (Alcoa 
F-20, Fisher Scientific Co., A-540), add 6 parts powd Mg(OH) 2 . 
Shake mixt. in screw-cap bottle until thoroly mixed. Add 8 
parts H 2 and immediately mix until all lumps disappear. 

(c) Phenylhydrazine soln. — Prep, immediately before use 
by dissolving 0.25 g phenylhydrazine. HO crystals in 25 mL 
H 2 0. Add 25 mL HCJ. 

(d) Methylbenzethonium hydroxide. —\M soln in MeOH (No. 
23, 394-3, Aldrich Chemical Co., Inc.). 

(e) Nifursol std solns. — (1) Stock soln. — 0.25 mg/mL. 
Weigh 25 mg nifursol (mp 224-226°) into 100 mL vol. flask. 
Add 5 mL DMF, mix until all material is dissolved, and dil. 
to vol. with MeOH. (2) Working soln. — 6.25 |xg/mL. Pi pet 
5.0 mL stock soln into 200 mL vol. flask, add 100 mL 95% 
DMF, and dil. to vol. with H 2 0. (Nifursol solns are not stable 
for long periods of time; prep, fresh as needed.) 

D. Extraction 

Weigh 5.00 g finely ground feed into 250 mL erlenmeyer. 
Add exactly 50 mL 95% DMF, stopper, and shake gently 5 
min on mech. shaker. Place sample 30 min in 60° H 2 bath, 
remove, and shake vigorously 30 min on mech. shaker. Filter 
thru rapid paper in buchner, using vac. Transfer 40.0 mL ali- 
quot of filtrate to beaker, add 40.0 mL H 2 0, stir, and let stand 
30 min in dark. 

E. Chromatography 

Add 7 cm specially prepd alumina to chromatgc tube contg 
glass wool plug. Tap column walls to settle alumina; then add 
1.5 cm Ottawa sand (No. S-15195, Sargent-Welch Scientific 
Co.). Prewash column with 50 mL 50% DMF just before use. 
Add DMF sample soln to column, discard first 60 mL eluate, 
and collect next 12 mL. (Decrease in flow rate may occur with 
some feed exts due to accumulation of fine ppt that settles on 
surface of alumina. Increase flow rate by stirring top of alu- 
mina and sand to break up ppt layer or by applying gentle air 
pressure to top of column.) 

F. Determination 

Pipet 5 mL aliquot into 20 mL test tube. Add 5 mL freshly 
prepd phenylhydrazine soln to tube, mix, and place 20 min in 
40° H 2 bath. Cool tube under cold tap H 2 (<20°) 5 min, 
add exactly 5 mL toluene, stopper tube, and shake vigorously. 
(Caution: Do not use black rubber stoppers.) Centrf. 5 min to 
clear toluene layer and transfer 3.0 mL toluene to 1 cm cell. 
Add 0.1 mL methylbenzethonium hydroxide to cell and mix 
immediately. Read sample within 1 min at 555 nm on spec- 
trophtr. Det. concn of nifursol from std curve. 

G. Preparation of Standard Curve 

Pipet 0, 2, 3, 4, and 5 mL aliquots working soln (equiv. to 
0.000, 0.0050, 0.0075, 0.0100, and 0.0125% in feed) into 
sep, 20 mL test tubes. Dil. to 5 mL with 50% DMF. Develop 
color as in 971. 46F and plot A against % drug in feed. 

Ref.: JAOAC 54, 66(197.1). 

CAS-16915-70-1 (nifursol) 



AOAC Official Methods of Analysis (1990) 



Nitarsone 



105 



961.25 Nihydrazone in Feeds 

Colorimetric Method 

First Action 1961 
Final Action 1962 

A. Reagents 

(a) 95% Dimethylformamide (DMF).— Dil. 95 parts DMF 
(Eastman Kodak Co. No. 5870, or equiv.) with 5 parts H 2 0. 
(Caution: See safety notes on dimethylformamide.) 

(b) Nihydrazone std solns. — (/) Stock soln. — Weigh 110 
mg cryst. nihydrazone (available from Norwich Eaton Phar- 
maceuticals Inc., 17 Eaton Ave, Norwich, NY 13815) into 
100 mL vol. flask, dissolve in DMF, and dil. to vol. with 
DMF. Protected from light, soln is stable several months. (2) 
Working soln.— For feeds contg 0.011% nihydrazone, pipet 1 
mL aliquot stock soln into 100 mL vol. flask, add 50 mL DMF, 
and dil. to vol. with H 2 0. 

B. Determination 

Weigh 10 g sample into 125 mL erlenmeyer, add exactly 
50 mL 95% DMF, stopper loosely, and place in boiling H 2 
bath 5 min (or until temp, of solv. reaches 90°). Shake mech. 
10 min and filter thru rapid paper. To 25 mL filtrate add 25 
mL H 2 and mix. Let stand, protected from light, >30 min. 
(Some solids may sep.; standing for longer time is permissi- 
ble.) 

Prep, ca 20 mm diam. adsorption column contg adsorbent, 
960.63A(e), to ht of 5 cm. (With highly colored feeds, use 
somewhat longer column.) Use plug of cotton or glass wool 
to support column, and similar plug or layer of washed sea 
sand on top. Pass sample soln thru column, collecting ca 15 
mL eluate. Pipet 5 mL aliquots into each of 2 tubes. Protect 
1 tube from light; to other add 3 drops freshly prepd 2% No. 
hydrosulfite soln, mix, and let stand 5 min. Treat 5 mL ali- 
quots dild std soln similarly. 

Pipet 5 mL phenylhydrazine soln, 960.63A(a), into all test 
tubes, mix, and heat 20 min in 40° H 2 bath. Cool by placing 
tubes in 15° H 2 bath 5 min. Add exactly 10 mL toluene to 
each tube, stopper, and shake vigorously 40 times. Sep. and 
centrf. toluene layer, and read A at 440 nm. 

% Nihydrazone 

L v* samp. ** red. samp./ 

X 0.011j/(A std - A redskl ) 
Ref.: J AOAC 44, 2(1.961). 
CAS-67-28-7 (nihydrazone) 



970.86 Nitarsone in Feeds 

Spectrophotometric Method 

First Action 1970 
Final Action 1973 

A. Principle 

Nitarsone is extd from feed with 50% dimethyls ulf oxide 
(DMSO) and sepd from interferences by alumina chromatgy. 
Nitro group is reduced with TiCl^ and resulting amine assayed 
colorimetrically at 530 nm with Bratton-Marshal! reaction. 
Arsanilic acid and carbarsone interfere. 

B. Reagents 

(a) Nitarsone std solns. — (7) Stock soln. — 1 mg/mL. Weigh 
100 mg nitarsone std (available from Salsbury Laboratories, 
Inc.) into 100 mL vol. flask and dil. to vol. with 4% NaOH. 
(2) Working soln. — 50 jjug/mL. Dil. 10 mL stock soln to 200 
mL with 4% NaOH. 

(b) Activated alumina. — Alcoa grade F-20, 80™ 200 mesh 
(available from Fisher Scientific Co. as Alumina, adsorption, 



Fisher No. A-540). To det. suitability of alumina, perform en- 
tire detn on 100 u,g nitarsone. Recovery should be >95%. 

(c) Dimethy [sulfoxide (DMSO) soln. — 50%. Dil. with equal 
vol. H 2 0. (Caution: DMSO can be harmful. Avoid skin con- 
tact by wearing heavy rubber gloves. Use effective fume re- 
moval device.) 

(d) Titanous chloride soln. — 4% aq. Prep, fresh daily from 
20% soln open <3 months and kept refrigerated, or from solid 
TiCl 3 . If >1 min is required for color disappearance in detn, 
use fresh source of TiCl 3 . (Caution: TiCl 3 is corrosive. Wear 
disposable plastic or rubber gloves. Avoid contact with eyes.) 

(e) Sodium nitrite soln. — 0.1% aq. Prep, weekly. 

C. Preparation of Standard Curve 

Pipet 0, 2, 5, 10, 15, 20, and 25 mL working soln into sep. 
100 mL vol. flasks and dil. to vol. with 4% NaOH. Pipet 10 
mL from each flask into sep. 50 mL vol. flasks, add \5 mL 
4% NaOH, and dil. to vol. with H 2 0. Pipet 4 mL from each 
flask into sep. test tubes and develop color as in 970. 86E, 
beginning "... add 2 drops 4% TiCl 3 ..." Std concns cor- 
respond to 0, 0.004, 0.010, 0.020, 0.030, 0.040, and 0.050% 
nitarsone in feeds. Plot std curve of A against % drug in feed. 

D. Preparation of Sample 

Accurately weigh 5 g finely ground feed into 100 mL vol. 
flask. Add 75 mL 50% DMSO, place sample on wrist action 
mech. shaker, and shake at room temp. 30 min. Dil. to vol. 
with 50% DMSO and mix. Transfer 30-40 mL to 50 mL centrf. 
tube and centrf. 10 min at 2000 rpm. 

E. Determination 

(Caution: See safety notes on pi pets.) 

Add alumina to 20 x 400 mm chromatgc tube with fritted 
glass disk to depth of 7 cm. Tap tube wall to settle alumina; 
then add 1 cm layer of sand. Prewash column with 50 mL 50% 
DMSO before use. 

Pipet 10 mL supernate from prepn of sample onto pre- 
washed column. For feeds contg >0.04% nitarsone, use smaller 
aliquot. Let sample enter column and then wash into column 
with several 5 mL portions H 2 0. Wash column with 75 mL 
H 2 and discard el u ate. 

Elute nitarsone with 65 mL 4% NaOH, discarding first 15 
mL. Collect remaining eluate in 100 mL vol. flask, letting 
column run dry. Nitarsone is eluted with ca 25-30 mL eluant. 
Dil. eluate to vol. with H 2 and mix. 

Pipet 4 mL dild eluate into 2 test tubes, add 2 drops 4% 
TiCl 3 to each with mixing, and shake or mix on Vortex mixer 
until black color disappears. Add 2 mL HC1 to each and mix 
thoroly. Add 0.5 mL 0.1% NaN0 2 , (e), and mix. After 5 min, 
add 0.5 mL 0.5% NH 4 sulfamate, 969.53A(c), and mix. After 
2 min, add 0.5 mL 0.1% coupling reagent, 969.53A(d), to 
one tube and 0.5 mL H 2 to second tube for blank. Let color 
develop 1 5 min; then read A of sample and blank at 530 nm 
on spectrophtr. Correct sample A for blank A and det. amt 
nitarsone in sample from std curve. 

Ref.: JAOAC 53, 641(1970). 

CAS-98-72-6 (nitarsone) 



960.65* Nithiazide in Feeds 

Spectrophotometric Method 

First Action 1960 

Final Action 1961 

Surplus 1989 



See 42.122-42.125, 14th ed. 



106 



Drugs in Feeds 



AOAC Official Methods of Analysis (1990) 



967.36* Nitrodan in Feeds 

Colorimetric Method 

First Action 1967 

Final Action 1968 

Surplus 1989 

(Not applicable in presence of interfering nitro compels) 
See 42.126-42.129, 14th ed. 



959.18 Nitromide in Feeds 

Spectrophotometric Method 
Final Action 

A. Reagents 

(a) Diethylamine reagent (DEA), aged, — (1 year or older.) 
Fresh DEA may be artifically aged as follows: Place 1 L DEA 
in dry 2 L flask with 40 g Na or K fluosilicate. Connect flask 
to 60 cm (24") bulb reflux condenser and reflux on sand bath 
2-3 days in hood. When reagent is sufficiently "aged," 2 mL 
clear DEA added to 8 mL dimethyl sulfoxide contg 50 fig 3,5- 
DNBA should develop max. color in ca 40 min. A as read on 
Beckman DU spectrophtr at 560 nm should be ca 0.375; on 
Klett-Summerson photoelec. colorimeter with No. 56 filter, ca 
200. Reagent must be free from turbidity. Prep, new std curve 
for each batch of DEA. 

(b) Nitromide std sains. — (/) Stock soln. — I mg/mL. Weigh 
100 mg nitromide (Salsbury Laboratories, Inc.) into 100 mL 
flask and dil. to vol. with MeOH. (2) Working soln. — 20 juig/ 
mL. Transfer 2.0 mL stock soln to 100 mL vol. flask and dil. 
to vol. with MeOH. 

B. Preparation of Standard Curve 

Place 1 .0, 2.0, 3.0, and 5.0 mL working soln contg 20, 40, 
60, and 100 fxg, resp., of nitromide in 4 colorimeter tubes. 
Evap. to dryness at 50° in air current. Dissolve residue in 8 
mL dimethyl sulfoxide at 70°, cool, and add 2 mL DEA re- 
agent. Place in dark at 20-25° and read after 1 hr. Plot std 
curve, using A as ordinate and concn as abscissa. 

C. Preparation of Sample 

Weigh 5.0 g feed, contg 0.025% nitromide, into 100 mL 
vol. flask and dil. to vol. with MeOH. Shake frequently 20 
min and let stand 40 min to permit feed particles to settle. 

If feed contains 0.075% nitromide, use 2 g finely ground 
feed; if 0.15%, use 1 g in 100 mL or 5 g in 500 mL MeOH. 
Prep, premixes by weighing appropriate sample and serially 
dilg MeOH ext. 

D. Determination 

Pipet 4 mL aliquot of ext into g-s test tube. Place tube in 
50° H 2 bath and evap. to dryness with air current directed 
onto surface of MeOH. Add 8 mL dimethyl sulfoxide and heat 
to 70° to hasten soln; cool, and add 2 mL DEA reagent. Place 
in dark 1 hr at 20- 25°. Det. A at 560 nm in Beckman DU 
spectrophtr, Klett-Summerson photoelec. colorimeter with No. 
56 filter, or similar instrument, against dimethyl sulfoxide as 
ref. 

Det. amt of nitromide in tube from std curve. 

% nitromide in feed 

= fxg nitromide in tube x 25 X J 00/5,000,000 

or |mg nitromide in tube x 5 = |xg nitro mide/g feed or ppm. 

Ref.: JAOAC42, 239(1959). 

CAS- 121-8 .1-3 (nitromide) 



952.29* Nitrophenide in Feeds 

Spectrophotometric Method 

Final Action 
Surplus 1980 

(Applicable in presence of arsanilic acid) 
See 42.120-42.122, 13th ed. 



967.37 Phenothiazine in Feeds 

Spectrophotometric Method 

First Action 1967 
Final Action 1970 

A. Reagent 

Phenothiazine std soln. — Dissolve 10 mg recrystd (from 10% 
soln in toluene) phenothiazine (1CI Americas, Inc., Eighty Four, 
PA 15330) in 50 mL alcohol and dil. to 100 mL with alcohol. 
For working stds, dil. with equal vol. of alcohol. (1 mL dild 
soln = 50 jxg phenothiazine.) Use freshly prepd soln; ale. solns 
gradually develop rose tint within few hr. 

B. Determination 

Place 1 g ground sample in 100 mL vol. flask, add 50 mL 
alcohol, and heat on steam bath 15 min. Cool, dil. to vol. with 
alcohol, mix, and let settle (ca 15 min) until supernate is clear. 

Place 2 mL aliquot in 25 mL vol. flask and add 10 mL 
alcohol. To flask add, in order given, 1 mL ./% ale. p-ami- 
nobenzoic acid soln, 1 mL aq. 2% NaN0 2 soln, and 1 mL 
HCI (1 + 3). Dil. to vol. with alcohol. Read A of green soln 
at 600 nm in spectrophtr against reagent blank. Det. amt of 
phenothiazine from std curve. 

% Phenothiazine — fxg/200 

Prep, std curve, using 1,2, and 3 mL dil. std soln, as above. 

Refs.: JAOAC 41, 338(1958); 42, 254(1959). 

CAS-92-84-2 (phenothiazine) 



967.38 Piperazine in Feeds 

Spectrophotometric Method 
Final Action 

A. Principle 

Piperazine or piperazine salt is quant, extd from feed into 
slightly acidic aq. soln. Dild filtrate is reacted with equal vol. 
benzoquinone soln at 80°. Colored complex formed is detd 
spectrophtric at 490 nm. 

Applicable to detn of 0.05-0.5% piperazine, usually present 
as one of its salts, in animal feeds. Amines give similar color 
reaction. Alkalies produce increased color; pH adjustment in 
method overcomes interference of this kind. 

B. Apparatus 

(a) Water hath. — Approx. 25 cm (10") diam. with 15-20 
cm (6-8") depth H 2 0. Thermostatically controlled at 80 ± 0.1°. 
(Viscosity bath is satisfactory.) 

(b) Test tubes.— Pyrex, 15 x 125 mm, with rubber stop- 
pers, and rack capable of supporting tubes when immersed in 
H 2 bath. 

C. Reagents 

(a) Qui none soln. — Dissolve 0.5 g p -benzoquinone in 2.5 
mL HO Ac and little alcohol in dry 100 mL vol. flask and dil. 
to vol. with alcohol. Keep soln in ice bath or refrigerator. Prep. 
fresh daily. (Caution: p -Benzoquinone is lachrymator; avoid 



AOAC Official Methods of Analysis (1990) 



Pyrantel Tartrate 107 



breathing vapor and contact with skin and clothing.) If blanks 
are high or variable, purify /?-benzoqui none by steam distn in 
hood. 

(b) Piperazine std solns. — (/) Stock soln. — Dissolve ex- 
actly 185 mg pure piperazine. 2HCJ (Salsbury Laboratories, Inc.) 
(equiv. to 100 mg piperazine) in H 2 and dil. to 250 mL. (2) 
Working soln. — 20 |xg/mL. Dil. 25.0 mL stock soln to vol. 
in 500 mL vol. flask. 

D. Preparation of Standard Curve 

Using working soln, add, by microburet or pipets, 20, 40, 
60, 80, and 100 |mg piperazine equiv. and intermediate values, 
if required, into test tubes. Dil. to 5 mL in each test tube with 
H 2 0. Include H 2 blank with each detn. 

Add 5 mL quinone reagent to each std and blank. Stopper 
tubes and mix by inverting. Remove stoppers and immerse in 
H 2 bath at 80 ± 0.1° exactly 10 min. (Bath temp, can be 
varied; use same temp, for samples and stds.) Immediately 
immerse tubes in ice bath 3 min. Let stand at room temp. >20 
min, but <40 min. Read A at 490 nm in 1.0 cm cells, using 
reagent blank to zero instrument. Plot A of each std against 
|xg piperazine. 

E. Determination 

Weigh 10.00 g well mixed feed (grind pellets in mortar) into 
500 mL (.1.6 oz) wide- mouth, screw-cap bottle. Add exactly 
200 mL H 2 from graduate and adjust to pH 4-5 (0.15 mL 
H 2 S0 4 (1 +2) is usually enough for 1 g feed). Cap or stopper 
bottle and place in wrist-action shaker 30 min. Add ca 5 g 
Celite (Note: Some grades may retain piperazine.) as slurry to 
buchner contg 9.0 cm Whatman No. 3 paper and pull down 
under full vac. Wash pad with small portion feed ext and dis- 
card washing. Rapidly filter remaining feed ext and reserve 
filtrate for color development. (Do not delay; turbidity may 
form.) 

Pipet 25.0 mL filtrate into 250 mL vol.. flask and dil. to vol. 
with H 2 0. Pipet 5 mL aliquot into test tube and immediately 
proceed with color development as in 967.38D. Include H 2 
blank with each detn. 

Prep, sample color blank for each feed as follows: Pipet 5 
mL aliquot dild ext into test tube and 5 mL H 2 into another 
test tube as ref. To each, add 5 mL soln contg 2.5 mL HO Ac 
dild to 100 mL with alcohol. Mix by inverting and omit heat- 
ing. Read A at same wavelength and subtract from sample 
reading. Include 1 or 2 stds with each detn to detect shift in 
std curve; adjust accordingly. Obtain |mg piperazine from std 
curve. 

% Piperazine = (juug piperazine X 10~~ 4 )/g sample in aliquot 

Ref.: JAOAC 50, 268(1967). 

CAS- 110-85-0 (piperazine) 



978.30 Pyrantel Tartrate in Feeds 

Spectrophotometric Method 

First Action 1978 
Final Action 1981 

(Applicable to range 0.0106-0.881 1%. Pyrantel tartrate solns 

are light sensitive. Exts must be protected from direct sunlight 

or artificial light.) 

A. Apparatus 

(a) High-speed blender.— Waring Blendor, or equiv, 

(b) Centrifuge. — International Model EXD (International 
Equipment Co.), or equiv. , equipped to hold 50 mL g-s centrf. 
tubes. 



(c) Filter aid. — Celite 545, acid- washed (Manville Filtra- 
tion and Minerals), or Millipore prefilter pad (No. AP2507500, 
Millipore Corp.), or equivs. 

(d) Filtrator. — Fisher No. 9-788, low form (Fisher Scien- 
tific Co.), or equiv. 

(e) Mixer. — Vortex Genie mixer (Scientific Products), or 
equiv. 

(f) Spectrophotometer. — For use at 31 I nm, with 1 cm cells. 

B. Reagents 

(a) Hydrochloric acid.— AN. Dil. 8.33 mL HC1 to 1 L 
with H 2 0. 

(b) Leaching soln. — Dissolve 100 g NaCl in 1 L H 2 0, add 
1 L MeOH, and mix vigorously. Prep, fresh daily. 

(c) Perchloric acid.~~Q.24M. Dil. 20 mL H'CI0 4 to 1 L 
with H 2 0. 

(d) Sodium hydroxide. — 0.17V. Dissolve 4 g NaOH in H 2 
and dil. to 1 L with H 2 0. 

(e) Pyrantel tartrate std solns. — (1) Stock soln. — 0.80 mg/ 
mL. Weigh 80.0 mg Pyrantel Tartrate Ref. Std (available from 
Pfizer, Inc., Quality Control, Agricultural Div., 1107 S Rt 
291, Lee's Summit, MO 64081) into 100 mL vol. flask, dis- 
solve in leaching soln, (b), and dil. to vol. with same solv. 
Use ultrasonic bath to speed soln. If std does not dissolve readily, 
remake. Prep, fresh daily. (2) Working soln.— 0A6 mg/mL. 
Pipet 20 mL stock soln into 100 mL vol. flask, dil. to vol. 
with leaching soln, and mix well. Prep, fresh daily. 

C. Preparation of Samples 

Weigh duplicate portions feed ground in high-speed blender, 
(a), into 125 mL erlenmeyers: 15.000 ± 0.010 g for 0.0106% 
pyrantel tartrate; 1.800 ± 0.010 g, 0.0881%; and 1.000 ± 
0.010 g, 0.881%. Add 60 mL leaching soln to 1 portion; add 
50 mL leaching soln and, by pipet, 10 mL working std soln, 
(e)(2), to other portion of 0.0106% or 0.0881% pairs or 10 
mL stock std soln, (e)(7), to other of 0.881% pair. Cap with 
polypropylene stopper and shake mech. 1 hr. 

Quant, transfer mixt. to buchner precoated with Celite or 
contg prefilter pad, (c), rinsing flask with small portions leach- 
ing soln, (b), and collecting filtrate under vac. with Filtrator, 
(d), contg 250 mL erlenmeyer, until vol. filtrate is 150 mL. 
Transfer filtrate to 200 mL vol. flask, and dil. to vol. with 
leaching soln. For 0.881% level, pipet 50 mL dild filtrate into 
250 mL vol. flask, and dil. to vol. with leaching soln. 

D. Determination 

Pipet 25 mL prepd sample soln into 250 mL separator contg 
5 g finely ground KI, and shake to dissolve. Pipet in 100 mL 
CHC1 3 and shake 45 sec. Transfer lower CHC1 3 layer to an- 
other 250 mL separator contg 25 mL 0.24M HC10 4 , and shake 
10 sec. Transfer ca 35 mL lower CHCI 3 layer to 50 mL g-s 
centrf. tube contg 10.0 mL 0.1N NaOH. Stopper tube, and 
mix on vortex mixer, (e), 15 sec while shaking back and forth. 
Centrf. 10 min at 990 x g (1800 rpm). Pipet 25 mL lower 
CHCI3 layer from tube into another 50 mL g-s centrf. tube 
contg 10.0 mL 0. \N HC1. Stopper tube, mix, and centrf. as 
before. Rapidly record A of upper aq. layer at 311 nm against 
CHCl r satd O.liV HC1 as ref. solv. 

% Pyrantel tartrate = (A/W) x \W /(A' - A)] x F x 100 

where A and A' refer to absorbance of sample and spiked sam- 
ple, resp.; W = g feed, supplement, or cone; W' = g pyrantel 
tartrate ref. std used to prep, std soln; F — [(1/100 mL) x 
(20 mL/100 mL) x 10 mL] for 0.0106% and 0.0881% levels; 
and F - [(1/100) x 10] for 0.881% level. 

Ref.: JAOAC 61, 296, 473(1978). 

CAS-3340 1-94-4 (pyrantel tartrate) 



108 



Drugs in Feeds 



AOAC Official Methods of Analysis (1990) 



966.27a Racephenicol in Feeds 

Final Action 1970 
Surplus 1975 

A. Method I— First Action 1966 

(Applicable to levels ^0.002%) 
See 42.128-42.131, 12th ed. 

B. Method ll—First Action 1968 

(Applicable to levels ^0.0005%) 
See 42.133-42.136, 12th ed. 



960.66* Reserpine in Feeds 

Photofluorometric Method 

First Action 1960 

Final Action 1964 

Surplus 1989 

(Applicable at 0.2-2.0 ppm level) 
5<?e 42.148-42.151, 14th ed. 



969.56* Ronnel in Feeds 

Gas Chromatographic Method 

First Action 1969 

Final Action 1970 

Surplus 1989 

See 42.152-42.154, 14th ed. 



970.87* Ronnel in Feeds 

Spectrophotometric Method 

First Action 1970 

Final Action 1972 

Surplus 1989 

(For mineral feed mixts contg 1-40% ronnel) 
See 42.155-42.159, 14th ed. 



971.47 Roxarsone in Feeds and Premixes 

Spectrophotometric Method 

First Action 1971 
Final Action 1974 

(Not applicable to pelleted feeds contg hemicellulose ext) 

A. Principle 

Roxarsone is extd from feed with 2% K 2 HP0 4 . Proteins are 
pptd at isoelec. point and removed by centrfg. Ext is treated 
with activated C at pH 12 to remove interferences and rox- 
arsone is detd spectrophtric at 410 nm. 

0. Apparatus 

(a) Centrifuge. —International Model V, or equiv. 

(b) Mechanical shaker. — Burrell wrist-action (Burrell Corp.), 
or equiv. 

C. Reagents 

(a) Potassium phosphate, dibasic, soln. — 2%. Dissolve 2 
gK 2 HP0 4 in 100 mL H 2 0. 



(b) Dilute hydrochloric acid. — Dil. 45 mL HC1 to 200 mL 
with H 2 0. 

(c) Sodium hydroxide soln. — Dissolve 24 g NaOH in 100 
mL H 2 0. 

(d) Charcoal adsorbent. — Activated (Darco G-60, or equiv.). 

(e) Roxarsone std soln. — 300 |mg/mL. Accurately weigh 300 
mg Roxarsone Ref. Std (Salsbury Laboratories) into 1 L vol. 
flask. Dissolve and dil. to vol. with 2% K 2 HP0 4 soln. 

D. Preparation of Sample 

Grind sample in high-speed blender to pass No. 20 sieve (ca 
3 min) and mix thoroly. Weigh 15.0 g ground sample into 125 
mL erlenmeyer, add 50.0 mL 2% K 2 HP0 4 soln, place on mech. 
shaker, and shake vigorously 5 min at room temp. Immedi- 
ately transfer to 100 mL centrf. tube and centrf. 10 min at 
3000 rpm. Perform extn and centrfg with min. delay. 

Decant 30 mL aliquot supernate into 50 mL graduated centrf. 
tube, pipet in 1 mL dil, HO, stopper, and mix thoroly. Let 
sample stand until protein flocculates (ca 15 min) and centrf. 
10 min at 3000 rpm. 

E. Purification 

Pipet 25 mL clear supernate into 125 mL erlenmeyer. Pipet 
in I mL NaOH soln, and mix thoroly. Add 2.0 g activated C 
and swirl sample several times during 30 min standing. Filter 
thru Whatman No. 42 fluted paper into 50 mL erlenmeyer. 
Repeat C treatment with second 2.0 g portion. 

F. Determination 

Place 3 mL filtrate in cell and det. A at 410 nm against H 2 
blank. Add 2 drops coned HO from dropper (or 5 mL serolog- 
ical pipet), mix, and reread ,4. Difference in A of acidic and 
basic samples represents roxarsone present. Det. amt from std 
curve. 

G. Preparation of Standard Curve 

Pipet 0, 1, 3, 5, 7, and 10 mL aliquots std roxarsone soln 
into 100 mL vol. flasks and dil. to vol. with 2% K 2 HP0 4 soln. 
Pipet 30 mL aliquot from each flask into sep. erlenmeyers, 
pipet 1 mL dil. HO into each flask, and mix. Pipet 25 mL 
aliquot this soln into sep. 50 mL erlenmeyers, pipet in 1 mL 
NaOH soln, and mix thoroly. Transfer 3 mL soln from erlen- 
meyers into sep. cells and det. A at 410 nm against H 2 blank. 
Add 2 drops coned HO from dropper (or 5 mL serological 
pipet), mix, and reread A. Plot difference in A of std solns 
against concn of solns expressed as 0, 0.001, 0.003, 0.005, 
0.007, and 0.010% roxarsone in feed when 15 g sample is 
used. 

H. Determination in Premix 

Weigh appropriate size sample of premix, place in 200 mL 
vol. flask, and add 50 mL 2% K 2 HP0 4 soln and 4.0 mL NaOH 
soln. Let stand 20 min, shaking occasionally; then dil. to vol. 
with 2% K 2 HP0 4 soln and let feed particles settle 30 min. 
Proceed as in 971.47D, par. 2, using appropriate aliquots and 
dilns to give final conens in range of std curve. 

(Example: 5% Premix. — Place 5 g thoroly mixed premix in 
200 mL vol. flask and ext as above. Transfer 10 mL aliquot 
ext to 100 mL vol. flask, dil. to vol. with H 2 0, and mix tho- 
roly. Transfer 10 mL aliquot this soln to another 100 mL vol. 
flask and dil. to vol. with H^O. Diln factor for this premix is 
1200.) 

After diln, transfer 30 mL aliquot to 125 mL erlenmeyer. 
Pipet in J mL dil. HO and mix. Transfer 25 mL this soln to 
125 mL erlenmeyer, pipet in I mL NaOH soln, mix thoroly, 
add 2.0 g activated C, shake several times during 30 min, and 
filter. Repeat C treatment. Read A of filtrate and det. concn 
of roxarsone as in 971.47F, multiplying by diln factor. 



AOAC Official Methods of Analysis (1990) 



Roxarsone 



109 



Ref.: JAOAC 54, 80(1971). 
CAS-121-19-7 (roxarsone) 



986.39 Roxarsone in Feeds 

Atomic Absorption Spectrophotometry Method 

First Action 1986 
Final Action 1989 

(Method dets total As and is not specific for roxarsone. Ap- 
plicable range is 0-50 ppm 4-hydroxy-3-nitrobenzene arsonic 
acid.) 

A. Principle 

Sample is extd with aq. ammonium carbonate soln and ana- 
lyzed by furnace AAS for total As content, which is converted 
by factor to roxarsone concn in finished feed. 

0. Reagents 

(a) Water. — Super Quality from Millipore Super Q system. 

(b) Nitric acid. — Mallinckrodt, ACS grade. 

(c) Argon. — Linde purified. 

(d) Nickel nitrate.— ~Ni(N0 2 ) 2 .6H 2 (Mallinckrodt AR). 

(e) Nickel nitrate soln. — Approximately 2000 ppm Ni. Dis- 
solve 10.0 g Ni(N0 3 ) 2 .6H 2 in H 2 and dil. to 1 L with H 2 0. 

(f) Ammonium carbonate. — Powder, purified (EM Science 
No. AX 1260). 

(g) Methanol.— Anhyd., ACS (EM Science No. MX0485). 
(h) Diluting soln. — Add 5 mL coned HN0 3 and 150 mL 

anhyd. MeOH to 1 L vol. flask, dil. to vol. with H 2 0, and 
mix. 

(i) Tantalum pentoxide. —99. 99% Ta 2 5 (Aldrich Chemi- 
cal Co.). 

(j) Tantalum pentoxide soln. — Suspend 2.0 g in 10 mL H 2 0. 

(k) Roxarsone std soln. — 1250 ppm roxarsone (356 ppm 
As). Accurately weigh 625.0 mg roxarsone ref. std (Salsbury 
Laboratories, Inc., 2000 Rockford Rd, Charles City, IA 50616) 
into 500 mL vol. flask. Dissolve and dil. to vol. with 2% am- 
monium carbonate soln. Caution: Wear protective clothing and 
avoid breathing dust. 

(1) Dilute roxarsone std soln. — 12.5 ppm roxarsone (3.56 
ppm As). Dil. 10.0 mL roxarsone std soln to 1 L with H 2 0. 

(m) Control feed extract. — Using typical nonmedicated 
poultry or swine ration, prep, feed ext as described under Sam- 
ple Preparation. Test suitability of control feed ext by dilg 1 
mL aliquot with dilg soln used in sample prepn. Set up AAS 
system and furnace conditions as described in procedure. It is 
not necessary to perform calibration for this test; absorbance 
reading is satisfactory. Zero spectrophtr on 20 jxL injection of 
dilg soln and measure A on 20 jxL control feed ext. Absor- 
bance reading <0.010 indicates suitability. 

(n) Working std soln. — Transfer 1.0 mL dil. roxarsone std 
soln to 10 mL vol. flask. Dil. to vol. with control feed ext. 
Transfer 1 .0 mL aliquot of this soln to 25 mL erlenmeyer and 
add 9.0 mL dilg soln. Twenty |ulL working std soln = 50 ppm 
roxarsone in feed sample for wts and vols used in procedure. 

C. Apparatus 

(a) Atomic absorption spectrophotometer. — Perkin-Elmer 
Model 5000, or equiv., with heated graphite atomizer furnace, 
autosampler, and printer sequencer. 

(b) Mechanical shaker. — Wrist-action. 

(c) Pipets.— Eppendorf: 10, 20, 50, and 1000 pJL. 

(d) Dispensing pipet. — Repipet (Labindustries, 620 Hearst 
Ave, Berkeley, CA 94710), or equiv., 10 mL capacity set to 
deliver 9.0 mL dilg soln. 



D. Sample Preparation 

Grind sample in Wiley mill to pass 20 mesh sieve and tho- 
roly mix. Weigh 5.0 g ground sample into 250 mL vol. flask 
or 300 mL erlenmeyer. Add 2.0 g ammonium carbonate pow- 
der and 200.0 mL H 2 0, place on mech. shaker, and shake 
vigorously 5 min at room temp. Remove flask from shaker 
and let suspended feed particles settle 15-30 min. Transfer 1.0 
mL aliquot of feed ext to 25 mL erlenmeyer and add 9.0 mL 
dilg soln. Mix thoroly. Repeat this step on reagent blank and 
on std-fortified control feed ext equiv. to 50 ppm roxarsone in 
feed. Samples and std are now ready for furnace AAS anal- 
ysis. 

£ AAS Conditions 

Set up graphite furnace and spectrophtr according to follow- 
ing conditions and allow 30 min warm-up time. Operating 
conditions: lamp, As EDL operated at 8 watts, properly aligned; 
lamp current, ma; wavelength, 193.7 nm; slit, 0.7 nm band- 
pass, low position; readtime, 5 s; mode, AA-BG; readout, concn; 
signal, peak ht on instrument display and A on recorder if used; 
std 1, SI — 50.0 ppm ((xg/g) roxarsone (use 3 digits), do not 
use S2 and S3. 

Install furnace assembly in AAS system and align as in man- 
ufacturer's instructions. 

F. Furnace Tube Coating Procedure 

Prep. 20% Ta 2 5 aq. suspension. Shake suspension vigor- 
ously, introduce 50 jxL aliquot into pyrolytically coated graph- 
ite furnace tube, and perform following sequence of opera- 
tions; H 2 flow, 1-2 L/min to cool furnace; Ar pressure, 35 
psi; on/off switch, on; gas control, on. 

Step 1 (drying): temp., 100°; ramp time, 10 s; hold time, 
90 s. 

Step 2 (charring): temp., 1000°; ramp time, 10 s; hold time, 
30 s. 

Step 3 (atomizing): temp., 2700°; ramp time, 5 s; hold time, 
10 s; stop flow, on. 

Repeat coating procedure twice (3 applications). Tube is now 
ready for furnace AAS use. 

(With initial coating, some material may flake after approx. 
35 firings. If this happens, pass small brush or Kimwipe thru 
tube to remove loose tantalum and apply single recoating.) 

G. Furnace Conditions for Sample Assay 

Step 1 (drying): temp., 100°; ramp time, 10 s; hold time, 
50 s. 

Step 2 (charring): temp., 1000°; ramp time, 10 s; hold time, 
30 s. 

Step 3 (atomizing): temp. , 2300°; ramp time, s; hold time, 
5 s; read, on; stop flow, on. 

Step 4 (burnout): temp., 2400°; ramp time, s; hold time, 
5 s; read, off; flow, 300 mL/min (stop flow, off). 

H. Autosampler Conditions 

Install autosampler assembly in furnace and align as per 
manufacturer's instructions. Operating conditions: power switch, 
on, and let autosampler go thru count down; program se- 
quence, press standby key to bring programmer into operating 
mode; method number, enter 1 and press method # key; re- 
calibrate, for full tray recalibrate at 9A, 1.8B, and 27C; last 
sample, program number for last sample vol. key; alternate 
vol., enter 10 uX and press alternate vol. key (Ni(N0 3 ) 2 .6H 2 
soln, 2000 ppm Ni); instrument program, enter 1 and press 
instrument program key; HGA program, enter 1 and press HGA 
program key. 

Use sample tray 1 with method 1 in autosampler sequence. 
This method uses external std technic for instrument calibra- 
tion. In this procedure, only position SI is used because cal- 



110 



Drugs in Feeds 



AOAC Official Methods of Analysis (1990) 



ibration is based on single point std with calibration std equiv. 
to 50.0 ppm roxarsone in finished feeds. 

/. Sample Analysis 

Load sample tray 1 as follows: Place blank of reagents (dilg 
soln (h)) in AZ location of tray; place 50.0 ppm roxarsone feed 
std in SI location and at position 1 (check sample), then load 
samples in sequence around tray, starting at position 2. Place 
2000 ppm Ni soln in reagent container and place in appropriate 
location for alternate sample in autosampler. Cover sample tray 
with cover provided to minimize evapn; these solns contain 
MeOH. 

Instrument and samples are now ready for calibration and 
sample analysis. Press start/stop key to start program in sam- 
pling cycle. AZ and SI calibration should be done in dupli- 
cate. Observed for duplicate SI values. These values should 
be within reasonable agreement (±5%) before program is al- 
lowed to proceed with samples. Instrument is recalibrated by 
autosampler in setup instructions which will monitor calibra- 
tion for any changes and update calibration as time progresses. 

J. Calculations 

Instrument is programmed to calc. sample ppm on basis of 
single point std equiv. to 50 ppm roxarsone. For wts or vols 
other than those specified, manual calcn is required. 

Ret.: IAOAC 69, 838(1986). 

CAS- 121 -19-7 (roxarsone) 



970.88 Sulfadimethoxine in Feeds 

Colorimetric Method 
First Action 1970 

A. Reagents and Apparatus 

See 969.53A(b), (c), and (d), and in addn: 

(a) Fie in soln. — 0.2%. Disperse 500 mg ficin (Calbio- 
chem, fig latex) in H 2 (preheated to 40°) and dil. to 250 mL. 
Use 10 mL of this warm soln in detn. {Caution: Ficin is very 
potent proteolytic enzyme which attacks living tissues. Avoid 
contact with skin and eyes and breathing dust.) (Ficin product 
listed is no longer available. Method may be satisfactory with- 
out ficin treatment, but users should verify recovery.) 

(b) Petroleum ether. — Bp 35-60°, purified on silica gel 
column. 

(c) Trichloroacetic acid soln. — 3%. Dissolve 30 g 
CCI3COOH in H 2 and dil. to 1 L. (Caution: See safety notes 
on trichloroacetic acid.) 

(d) Sulfadimethoxine std soln. — Accurately weigh 125 mg 
sulfadimethoxine USP Ref. Std and transfer quant, to 100 mL 
vol. flask. Add ca 70 mL acetone and shake until completely 
dissolved. Dil. to vol. with acetone and mix. Pipet 20 mL soln 
into 200 mL vol. flask, dil. to vol. with acetone, and mix. 
Pipet 10 mL (or 5 mL if working at 0.00625% level) of last 
diln into 200 mL vol. flask contg 10 mL 0.2% ficin soln. Add 
ca 120 mL acetone and 2 mL 40% NaOH soJn, and mix. Dil. 
to vol. with acetone and mix. Pipet 25 mL final diln into 50 
mL g-s centrf. tube, evap. almost to dryness under N stream 
in 50° H 2 bath, and proceed as in 970.88B, beginning "Pipet 
15 mL pet ether into centrf. tube ..." Resulting clear filtrate 
is std soln. 

(e) Reagent blank. — Into 200 mL vol. flask pipet 10 mL 
0.2% ficin soln and 2 mL 40% NaOH soln. Dil. to vol. with 
acetone and mix. Pipet 25 mL of this soln into 50 mL g-s 
centrf. tube, evap. almost to dryness under N stream in 50° 
H 2 bath, and proceed as in 970. 88B, beginning "Pipet 15 
mL pet ether into centrf. tube ..." Resulting clear filtrate is 
blank soln. 



(f) Spectrophotometer . — With 5 cm cells, or photoelec. 
colorimeter with 540 nm filter, or equiv. 

B. Preparation of Sample 

(Caution: See safety notes on blenders, pipets, flammable 
solvents, and acetone.) 

Pipet 10 mL 0.2% ficin soln into high-speed blender. Ac- 
curately weigh 10 g sample into blender, spreading carefully 
on surface of liq. Let sample soak 10 min. 

Add ca 120 mL acetone. Blend 2 min, adjusting speed with 
variable transformer, so that acetone does not wet screw cap. 
(Note: To release pressure, stop blending after 3-4 sec and 
unscrew cap momentarily.) Blend 2 min and remove screw 
cap. Push down into acetone all solid particles adhering to con- 
tainer wall, using rubber policeman. Replace screw cap and 
continue blending 1 min. Remove screw cap, pipet 2 mL 40% 
NaOH soln into container, and continue blending 2 min. Push 
down into acetone all solid particles adhering to wall of con- 
tainer, using rubber policeman. 

Quant, transfer blender contents to 250 mL g-s graduate, 
using small portions acetone to total vol. of 200 mL. Stopper, 
mix well, and let liq. and solids sep. Wrap tip of 50 mL pipet 
with glass wool and transfer ca 40 mL ext to 50 mL g-s centrf. 
tube. Centrf. 5 min at 2000 rpm. Pipet 25 mL clear acetone 
ext into another centrf. tube and evap. almost to dryness (only 
few drops of oily, sirupy liq. left) under N stream in 50° H 2 
bath. Pipet 15 mL pet ether into centrf. tube and dissolve or 
disperse residue in it, 

Pipet 25 mL 0.2jV NaOH into centrf. tube, stopper, and shake 
mech. 5 min. Centrf. 10 min at 2000 rpm. By pipet, transfer 
lower NaOH layer (ca 24 mL) into another centrf. tube and 
centrf. 10 min at 2000 rpm. Pipet 20 mL clear soln into 100 
mL vol. flask. Dil. to vol. with 3% CCl 3 COOH soln. Mix and 
let stand 10 min. Filter entire soln thru Whatman No. 42 pa- 
per, discarding first 10 mL filtrate. If turbid, filter thru second 
paper. Clear filtrate is sample soln. 

C. Determination 

(a) Reading on spectrophotometer with 5 cm cells. — Pipet 
following vols (mL) of indicated solns into 6 sep. labeled 25 
mL vol. flasks: 



Soln 


Sample 

1 


Sample 
2 


Sample 
Blank 


Std 1 


Std 2 


Reagent 

Blank 


Sample 

Std 

Blank 


15 


15 


15 


15 


15 


15 



Pipet 1 mL 0.1% NaN0 2 soln, 969.53A(b), into each, mix, 
and let stand 3 min. Pipet 1 mL 0.5% NH 4 suifamate soln, 
969.53A(c), into each, mix, and Jet stand 2 min. Pipet 1 mL 
0.1% coupling reagent, 969.53A(d), into all. except sample 
blank, and 1 mL H 2 into sample blank. Mix and let stand 10 
min in dark. Dil. each flask to vol. with H?0 and mix. Mea- 
sure A of each soln at 540 nm in 5 cm cell against reagent 
blank in ref. cell. 

(b) Reading on photoelectric colorimeter . — Proceed as in 
(a) except do not dil. after standing, but read at existing vol.; 
50 mL g-s centrf. tubes may be used in place of vol. flasks. 
Transfer solns from flasks or tubes to matched colorimeter tubes. 
Set instrument with 540 nm filter to 100% T (0 A) with tube 
contg reagent blank. Det. A (= 2 - log (%T)) of each of other 
tubes contg samples, sample blank, and stds. 

(c) Calculation. — Higher levels of sulfadimethoxine: 

% Sulfadimethoxine = [(A - A ) X S]/(1000 x A' X W) 



AOAC Official Methods of Analysis (1990) 



Sulfamethazine 



111 



Lower levels of sulfadimethoxine: 

% Sulfadimethoxine = [(A - A ) x S]/(2000 X A' x W) 

where A, A (h and A' refer to sample, reagent blank, and std, 
resp.; W = g original sample; and S - mg std weighed. 

Ref.: JAOAC 53, 638(1970); 72, 106(1989). 

CAS- 122- 11-2 (sulfadimethoxine) 



951.07* Sulfaguanidine in Feeds 

Spectrophotometric Method 

First Action 
Surplus 1989 

See 42 .171, 14th ed. 

969.57 Sulfamethazine in Feeds 

Spectrophotometric Method 
First Action 1969 

(Applicable to feeds contg procaine penicillin. Not applicable 
to feeds made from granule-stabilized Tylan-Sulfa premixes.) 

A. Reagents 

See 969.53A(b), (c), and (d) and in addn; 

(a) 50% Methanol sola.— 50% (v/v) aq. soln of MeOH. 

(b) Sulfamethazine std so Ins . — ( / ) Stock soln . — Accurate! y 
weigh 0.100 g pure sulfamethazine (available from American 
Cyanamid Co.) into 100 mL vol. flask. Add 50 mL 50% MeOH 
soln and shake until dissolved. Dil. to vol. with 50% MeOH 
soln. Soln is stable at least several weeks. (2) Intermediate 
soln. — Pipet 5 mL stock soln into 200 mL vol. flask, dil. to 
vol. with 50% MeOH, and mix well. Soln is also stable sev- 
eral weeks. (3) Working soln. — 2.5 |ULg/mL. Pipet 10 mL in- 
termediate soln into 100 mL vol. flask, add 1 mL HC1 and 50 
mL 50% MeOH, dil. to vol. with H 2 0, and mix well. Soln is 
stable ca 2 weeks. 

B. Preparation of Sample 

Weigh 5.00 g sample into 250 mL g-s erlenmeyer. Add 100.0 
mL 50% MeOH soln, shake well on mech. shaker 1 hr, and 
centrf. Pipet aliquot supernate contg ca 250 |ig sulfamethazine 
into 100 mL vol. flask, add 50% MeOH, if necessary, to vol. 
of ca 60 mL, followed by 1.0 mL HC1, and 10 mL 1% ZnS0 4 
soln. Let stand 10 min, dil. to vol. with H 2 0, and mix. 

C. Determination 

Filter portion of prepd soln thru Whatman No. 42 paper, or 
equiv., into 250 mL flask. Filtrate should be clear. Pipet two 
10 mL aliquots filtrate and 10 mL working std soln into sep. 
50 mL centrf. tubes. To each tube add 1.0 mL 0.1% NaN0 2 
soln; mix and let stand 3 min. Add 1.0 mL 0.5% NH 4 sul- 
famate soln; mix and let stand 2 min. Add 1.0 mL 0.1% N- 
(l-naphthyl)ethylenediamine.2HCI soln to one of sample solns 
and to std soln. To second sample soln add 1.0 mL H 2 (sam- 
ple blank). Mix all solns well and let stand 10 min. 

To sample, sample blank, and std soln add ca 10 mL CHCl 3 , 
stopper, and shake vigorously 30 sec (30 sec is required to 
ensure complete removal of procaine dye). Add 0.8 mL ION 
NaOH to sample, sample blank, and std soln. Stopper and shake 
vigorously >1 min to ensure complete removal of procaine 
dye. Centrf. solns at 2000 rpm 5 min or until aq. layer is com- 
pletely clear. Remove 10.0 mL aq. phase with pipet and trans- 
fer to 50 mL erlenmeyer or 50 mL beaker. Add 1.0 mL HO 
and remove fumes formed in flask with aspirator or air stream. 



Read A of sample, sample blank, and std (A') at 540 nm in 
spectrophtr, against H 2 blank. Correct A of sample by sub- 
tracting that of sample blank. 

% Sulfamethazine = (A /A') X (2,5 fxg/mL) 

x (100 mL/mL ext aliquot taken) x (100 mL/5 g) 

x (l g/10 6 p,g) x 100 

Refs.: JAOAC 51, 1282(1968); 72, 106(1989). 

CAS-57-68-1 (sulfamethazine) 



963.34 Sulfanitran in Feeds 

Spectrophotometric Method 
Final Action 

A. Reagents 

See 969.53 A(b), (c), and (d) and in addn: 

Sulfanitran std solns. — (./) Stock soln. — 100 jxg/mL. Ac- 
curately weigh 100 mg pure sulfanitran (available from Sals- 
bury Laboratories, Inc.) into 1 L vol. flask, add enough IN 
NaOH for complete soln, and dil. to vol. with H 2 0. (2) Work- 
ing soln. — 10 |xg/mL. Dil. 10 mL stock soln to 100 mL with 
H 2 0. 

B. Preparation of Standard Curve 

Pipet 0, 4, 6, 8, and 10 mL aliquots working std soln into 
sep. 50 mL vol. flasks. Add 0.5 mL HO and adjust vol. with 
H 2 to ca 15 mL. Place flasks in boiling H 2 bath I hr to 
deacetylate. Cool, and dil. to \o\. Transfer 5 mL aliquot from 
each flask to sep. colorimeter tubes. Develop color by adding 
0.5 mL 0.1% NaN0 2 soln (not >5 days old, stored in refrig- 
erator), 0.5 mL 0.5% NH 4 sulfamate soln, and 0.5 mL cou- 
pling reagent. Det. A at 540 nm against reagent blank (0 mL 
aliquot). 

(To establish most reliable std curve, make detns on 3 sep. 
days and use av. values.) 

C. Extraction and Deacetylation 

(For premixes, use proper dilns to give 5-10 (xg sulfanitran in 
final aliquot, taking dilns into consideration in final calcn.) 

Weigh 5.0 g sample into 100 mL vol. flask and add 80 mL 
MeOH. Place flask in 60° H 2 bath until MeOH is hot. Re- 
peatedly remove and immerse flask during 20 min, shaking 
frequently. Cool to room temp., and dil. to vol. with MeOH. 
Shake thoroly, and let stand 40 min to permit particles to set- 
tle. 

Pipet 25 mL aliquot MeOH ext into 50 mL vol. flask. Add 
10 mL H 2 0, 5 mL 7 .0% ZnS0 4 soln, and ca 3 drops IN NaOH 
to improve ilocculation. (Keep near neutrality.) Place flask in 
boiling H 2 bath 2 min to aid pptn; then cool to room temp., 
dil. to vol., mix thoroly, and filter thru Whatman No. 42 pa- 
per, or equiv. Discard first 5 mL filtrate. 

Pipet 10 mL aliquot filtrate into 50 mL vol. flask contg 8.0 
mL H 2 and 0.5 mL HO. Place flask in boiling H 2 bath 1 
hr to evap. MeOH and deacetylate sulfanitran, shaking fre- 
quently during first 15 min. Cool to room temp, and dil. to 
vol. with H 2 0. Centrf. if turbidity appears. 

D. Determination 

Place 5.0 mL aliquot in each of 2 colorimeter tubes. To I 
tube (blank) add 1.0 mL H 2 and 0.5 mL coupling reagent. 
To other tube add 0.5 mL 0. 1 % NaN0 2 soln; after 3 min, add 
0.5 mL 0.5% NH 4 sulfamate soln, wait 2 min, and add 0.5 
mL coupling reagent. Close tube with thumb and invert im- 
mediately after adding each reagent. Let stand 10 min for color 
development and det. A of sample and blank at 540 nm in 



112 



Drugs in Feeds 



AOAC Official Methods of Analysis (1990) 



spectrophtr or colorimeter against H 2 0. Det. amt sulfanitran 
from std curve after subtracting A of blank. 

% sulfanitran in sample = (xg sulfanitran in tube 
x 200 x 100/ (5,000,000 x Lig sample) 

= u,g sulfanitran x 0.004 

Ref.: J AOAC 46, 452(1963). 

CAS- 122- 16-7 (sulfanitran) 



963.35 Sulfaquinoxaline in Feeds 

Spectrophotometry Method 

First Action 1973 

Final Action 1988 

Method I 

(Applicable only to nonpelleted feeds contg arsanilic acid. In 
absence of arsanilic acid, use 963.35F>) 

A. Principle 

Sulfaquinoxaline is extd from feed with DMF and sepd from 
interfering substances by column chromatgy on alumina. Iso- 
lated sulfaquinoxaline is acidified, diazotized, and coupled in 
presence of Zr, and colored complex is extd with BuOH and 
measured at 550 nm. Arsanilic acid remains in final aq. soln 
and can be measured at 540 nm and compared with std treated 
similarly. 

B. Reagents 

See 969.53A(b), (c), and (d) and in addn: 

(a) Alkaline salt soln. — Dissolve 2.0 g NaOH and 100.0 g 
NaCl in 500 mL H 2 0. 

(b) Zirconium soln. —Dissolve 5.0 g zirconyl chloride, 
ZrOCl 2 .8H 2 (Fisher Scientific Co.), in 100 mL H 2 0. 

(c) Sulfaquinoxaline std solns . — (7 ) Stock soln . — Weigh 40 . 
mg Sulfaquinoxaline Ref. Std (available from Merck & Co., 
Inc.) and dissolve in 50.0 mL DMF. Soln is stable at least 1 
month if kept tightly stoppered and protected from light. (2) 
Intermediate soln. — 80 |xg/mL. Dil. 5 mL stock soln to 50 
mL with DMF. (3) Working soln. — 8 |xg/mL. Dil. 5 mL in- 
termediate soln to 50 mL with DMF. Prep, from freshly prepd 
intermediate soln just before use. 

(d) Butanol mixture. — Mix 100 mL n-hexane with 400 mL 
h-BuOH. 

C. Preparation of Sample 

Weigh 4.00 g ground feed sample into 100 mL vol. flask. 
Add 50.0 mL DMF, stopper, and agitate by mag. stirrer or 
mech. shaker 60 min. Transfer mixt. to 50 mL centrf, tube 
and centrf. 5 min at 2500 rpm. 

D. Chromatography 

(a) Preparation of column. — Constrict end of 50-60 cm 
length of 9-11 mm id glass tubing by rotating in hot flame 
until opening is 4-5 mm. Insert small plug of Pyrex glass wool 
in lower end and compress with glass rod to thickness of 2- 
3 mm. Transfer 5.0 g alumina, 961.24B(b), to dry tube and 
pack by gentle tapping while applying vac. 

(b) Separation. — Pipet 10 mL clear ext onto column and 
let pass thru by gravity. Do not let column run dry; keep 5 
mm head of liq. Wash inner walls with two 5.0 mL portions 
CHC1 3 . Let final washing drain until no further liq. appears at 
tip. Discard effluent and washings. Attach column tip to vac. 
and draw air thru until alumina is dry, indicated by tube re- 
turning to room temp. Elute column by gravity with 25 mL 
alk. salt soln, collecting eluate in 25 mL vol. flask. Add 1.0 
mL HC1 to eluate, dil. to vol. with H 2 0, and mix well. 



Prep, reagent blank by transferring 10 mL DMF onto fresh 
column and proceeding as for sample. Prep, std by transferring 
10.0 mL sulfaquinoxaline working std soln onto fresh column 
and proceeding as for sample. 

E. Determination 

Transfer 10 mL aliquots of each eluate to sep. centrf. tubes. 
Add 2.0 mL Zr soln and mix. Add 1.0 mL 0.1% NoN0 2 soln, 
mix, and let stand 2 min. Add 1.0 mL 0,5% NH 4 sulfamate 
soln, mix, and let stand 2 min. Add 1 .0 mL coupling reagent, 
969.53A(d), mix, and let stand 10 min. Add 2.0 g NaCl and 
10.0 mL BuOH mixt., stopper, and shake vigorously until NaCl 
dissolves. Centrf., carefully transfer portion of clear, colored 
top solv. layer to 1 cm cell, and read A at 550 nm against 
BuOH mixt. Correct for reagent blank. 

% Sulfaquinoxaline = 0.04 x (A/A')/W 

where A and A' refer to sample and std (blank corrected), resp. , 
and W = g sample. 



Method II— Final Action 1960 

(Applicable in absence of arsanilic acid) 

F. Determination 

Weigh 5 g ground sample into 250 mL vol. flask, add 150 
mL H 2 and 5 mL 0.5N NaOH, and place in boiling H 2 bath 
15 min. Remove, cool, dil. to vol. with H 2 0, mix, and let 
settle. Transfer 50 mL supernatant to 100 mL vol. flask, add 
3 mL HC1, and dil. to vol. Mix, and filter thru 18.5 cm What- 
man No. 2 paper (or equiv.), discarding first 15 mL filtrate if 
turbid. 

To 10 mL filtrate in each of two 50 mL beakers add 2 mL 
freshly prepd 0.1% NaN0 2 soln and let stand 3 min. Add 2 
mL 0.5% NH 4 sulfamate soln and let stand 2 min. Add 1 mL 
coupling reagent, 969.53A(d), to first beaker and 1 mL H 2 
to second beaker. Mix thoroly after adding each reagent. After 
10 min, read A in spectrophtr at 545 nm. Subtract A of feed 
blank from sample A and det. amt of sulfaquinoxaline from 
std curve. Divide by 1000 to obtain % sulfaquinoxaline. 

Prep, std curve as follows: Dissolve 0.250 g pure sulfaquin- 
oxaline in 5 mL 0.5N NaOH and 50 mL H 2 in 500 mL vol. 
flask, and dil. to vol. with H 2 0. Pipet 5 mL aliquot of this 
soln into 100 mL vol. flask and dil. to vol. with H 2 0. Pipet 
2, 4, 6, 8, and 10 mL portions of this dild soln (equiv. to 50, 
100, 150, 200, and 250 |xg sulfaquinoxaline, resp.) into sep. 
100 mL vol. flasks, add 3 mL HC1 to each flask, and dil. to 
vol. with H 2 0. Treat 10 mL aliquots of these final dilns as in 
second par. Det. A at 545 nm against H 2 blank, and plot A 
against u,g sulfaquinoxaline. 

Refs.: JAOAC 33, 156(1950); 38, 229(1955); 39, 307(1956); 
56, 758(1973); 59, 399(1976); 62, 423(1979). 

CAS-59-40-5 (sulfaquinoxaline) 



974.46 Sulfonamides in Feeds 

Spectrophotometric Method 
First Action 1974 

(Applicable to premixes and cones) 

A. Determination of Absorptivities 

Prep. sep. std solns of sulfathiazole (SZ), sulfamerazine (SM), 
and sulfamethazine (SH) by accurately weighing ca 50 mg each 



AOAC Official Methods of Analysis (1990) 



Sulfonamides 



113 



compd and transferring to sep. 50 mL vol. flasks; add 5 mL 
alcohol and 2 mL NH 4 OH, and swirl to dissolve. For 
sulfaquinoxaline (SQ), weigh 45 mg, warm on steam bath to 
dissolve, and cool. Dil. each flask to vol. with alcohol, and 
mix. Eva p. 2 mL aliquots to dryness in sep. small beakers, 
transfer to sep. 200 mL vol. flasks (100 mL for SQ) with sev- 
eral small portions QAN NaOH, and dil. to vol. with 0.1/V 
NaOH. Also dil. 25 mL aliquot SQ soln to 50 mL with 0.17V 
NaOH. Obtain spectrum of each soln in 1 cm cell against 0.17V 
NaOH from 400 to 220 nm. 

Calc. a 255 (1.00 mg/100 mL) = A X 50/W 

where A is reading at max., ca 255 nm, corrected for A at 400 
nm, if any, and W is mg compd weighed. (For SQ, use A of 
dild soln.) 

For SQ, also calc. a 35S (2.00 mg/100 mL) -Ax 50/VV, 
where A is reading at max., ca 358 nm, corrected for A at 400 
nm, if any, of more coned soln. 

B. Preparation of Sample 

(a) Solids. — Transfer accurately weighed sample contg ca 
50 mg sulfonamide with lowest concn to 50 mL vol. flask, 
add 5 mL alcohol and 2 mL NH 4 OH, and warm 10 min on 
steam bath. Cool to room temp., dil. to vol. with alcohol, and 
mix. 

(b) Liquid concentrates. — Pipet aliquot contg ca 200 mg 
sulfonamide with lowest concn into 200 mL vol. flask, dil. to 
vol. with alcohol, and mix. 

For each sulfonamide declared, calc. an R value to 2 dec- 
imal places by dividing its labeled amt by that of sulfonamide 
with lowest labeled amt, whose R = 1.00. Calc. to 2 decimal 
places. Designate each ratio as R S q, R S z, ^sm> an ^ ^sh, and 
their sum as R T . (If 4 sulfonamides are present in equal amts, 
all/? - 1.00 and/? T = 4.00.) 

C. Determination of Sulfaquinoxaline 

Pipet 5 mL sample soln into g-s flask and add accurately 
measured vol. alcohol so that total mL of final soln = 10 x 
R T (Soln f). Mix, pipet 10 mL into small beaker, and evap. to 
dryness on steam bath. Transfer residue to 100 mL vol. flask 
with several small portions 0.1 Af NaOH, dil. to vol. with 0. \N 
NaOH, and mix (Soln If). Obtain spectrum from 400 to 300 
nm, and det. A at max., ca 358 nm. 

% SQ = [A 35g x 2 x R T x (V/5) x 100]/(a 358 X S) 

where V = mL original sample soln (50 or 200). and S — mg 
original sample (for solids) or mL x 1000 (for liqs). 
If Na salt declared, NaSQ - SQ/0.9318. 

D. Determination of Total Sulfonamides 

Dil. 20.0 mL Soln U to 100 mL with 0.17V NaOH. Obtain 
spectrum from 400 to 230 nm and det. A T at max. , ca 255 nm. 

% Total sulfonamides - [A r X V x R T 2 x 100]/ 
l(R x a )sQ H- (R X a ) sz + (R x a ) SM 

+ (R x a ) SH \ X S 

If Na salts are present, multiply each a by appropriate factor: 
NaSQ, 0.9318; NaSZ, 0.9207; NaSM, 0.9232; and NaSH, 
0.9268. 

Ref.: JAOAC 57, 345(1974). 

CAS- 127-79-7 (sulfamerazine) 
CAS-57-68-1 (sulfamethazine) 
CAS-59-40-5 (sulfaquinoxaline) 
CAS-72-14-0 (sulfathiazole) 



974.47 Sulfonamides in Feeds 

Thin Layer Chromatographic Method 
First Action 



A. Preparation of Plates 

(a) Plates A.— Weigh 30 g silica gel H or HF 254 (Brink- 
mann Instruments, Inc.) into 250 mL g-s flask. (Add 100 mg 
fluorescent indicator H 254 if silica gel H is used.) Add 70 
mL H 2 and shake well 1 min. Coat five 20X20 cm plates 
with 0.25 mm layer and air dry. Do not dry in oven and do 
not store in presence of drying agent. 

(b) Plates B. — Proceed as in (a) but use 0AN NaOH in- 
stead of H 2 to slurry silica gel. 

B. Preparation of Blanks 

Develop a plate A in CHCl 3 -MeOH (97 + 3) to ht of ca 15 
cm. Scrape 2 spots, each ca 2 sq cm, from developed section 
of plate, into centrf. tubes and ext with 10 mL 0AN NaOH. 
Scrape 2 addnl spots, each ca 4 sq cm, from developed portion 
and ext with 50 mL 0.UV NaOH. Centrf. the 4 exts 5 min at 
high speed and decant most of soln into sep. small beakers, 
being careful not to disturb sediment. 

Develop a plate B in CH'CJs-M'eOH (90 + 10) and proceed 
as above. 

Det. A at max. , ca 255 nm, of all 8 exts. A should be <0.04. 
If readings are low and reproducible, average each set of 4 
values for 2 and 4 sq cm exts, resp. If readings are high or 
not reproducible, recentrf. exts at higher speed, and be very 
careful to exclude sediment during decanting. Av. blank A may 
be used for all detns which use same batch of silica gel and 
same speed of centrfg. 

C. Thin Layer Chromatography 

Spot 100 (xL sample soln, 974. 46B, on plate A and plate B 
by repeated application of adjacent drops on line 4 cm long, 
drying each drop with gentle air current before applying an- 
other drop at same place. Develop plate A in CHCl 3 -MeOH 
(93 + 7) and plate B in (90 4- 10) until sol v. front reaches 
top of plate. 

View plates under shortwave UV light and delineate each 
spot with dissecting needle, including small margins whenever 
possible. Plate A should have 2 completely sepd spots, des- 
ignated as c (lower: SZ) and d (upper: SM + SH + SQ); plate 
B should have 3 completely sepd spots designated as /(lower: 
SQ + SZ), h (middle: SM), and p (top: SH). If less than in- 
dicated no. of spots are sepd, spot another aliquot over slightly 
longer line and develop as before. 

Use collection tube consisting of 7-8 cm of 8 mm od glass 
tubing with short constriction at one end and medium fritted 
glass disk fused near center. Clean tube with strong air current 
and attach wide tube to vac. Draw as much of spot as possible 
into tube, using narrow tube to loosen adsorbent layer. With- 
out disconnecting vac, transfer tube to 10 mL vol. flask for 
spot c and 50 mL for spot d. Release vac, and transfer ma- 
terial to flask with repeated gentle tapping. Repeat until entire 
spot has been transferred; then blow out tube into flask with 
gentle air current to remove last of particles. 

Fill each flask ca V2 full with 0. \N NaOH, swirl well 1 min, 
dil. to vol. with 0AN NaOH, and mix. Centrf. all or >10 mL 
each soln at same speed used for blanks and carefully decant 
ca 8 mL into small beakers. Obtain spectrum of each soln from 
400 to 230 nm and record A c of spot c ext and A$ of spot d 
ext at max., ca 255 nm. Correct A c for av. blank of 2 sq cm 
exts and A d for av. blank of 4 sq cm exts. 



114 



Drugs in Feeds 



AOAC Official Methods of Analysis (1990) 



Calc. recovery factor, F = f(5 X A d ) + A C \/(R T X A T ) 

% SZ - (A c x V x 100)/(a sz ,255 x F x S) 

% NaSZ = % SZ/0.9207 

From plate B, transfer spot f to 50 mL vol. flask and spot 
h and p to 10 mL vol. flasks. Obtain blank corrected A h A h , 
and v4 p as for exts of plate A. 

Calc. recovery factor F f - [(5 X A f ) + A h + A P ]/(J? T x A T ) 

% SM - (A h X V x 100)/(a SM x F' x S) 

% SH - 04 p x V x 100)/(a SH x F' x S) 

% NaSM = % SM/0.9232 

% NaSH = % SH/0.9268 

Ref.: J AOAC 57, 345(1974). 

CAS- 127-79-7 (sulfamerazine) 
CAS-57-68-1 (sulfamethazine) 
CAS-59-40-5 (sulfaquinoxaline) 
CAS-72-14-0 (sulfathiazole) 



966.28 Thiabendazole in Feeds 

Spectrophotometric Method 

First Action 1966 
Final Action 1967 

Method I 

(Applicable to all feeds) 

A. Principle 

Thiabendazole is extd from feed with 0A.N HC1. Interfer- 
ences are removed by adjusting ext to pH 5-6 with Na citrate 
and extg with CHCl 3 . Thiabendazole is re-extd with 0.1N HCl 
and reduced with Zn slurry in 30% glycerol in presence of 
p-phenylenediamine. Oxidn with ferric iron yields blue com- 
plex which is extd with BuOH and measured at 605 nm. 

B. Reagents 

(a) Zinc dust. — Reagent grade. Crush fine lumps with spat- 
ula immediately before use. 

(b) Zinc slurry. — Weigh 50 mg /?-phenylenediamine.2HCl 
(Caution: /?-Phenylenediamine may be harmful; see safety notes 
on toxic dusts.) and 2 g Zn dust into dry 100 mL g-s graduate. 
Add 100 mL 30% (v/v) glycerol soln, stopper, and shake ca 
30 sec to suspend Zn dust uniformly. (There must be no ag- 
glomeration of Zn.) Prep, just before use and use immediately. 

(c) Ferric soln.— Dissolve 15.0 g FeNH 4 (S0 4 )?.12H 2 in 
75 mL H 2 0, add 10.0 mL \N H 2 S0 4 , dil. to 100 mL, and 
mix. 

(d) Thiabendazole std solns. — (J) Stock soln. — 0.5 mg/mL. 
Dissolve 50.0 mg Thiabendazole Ref. Std (available from Merck 
& Co.) in 0.1JV HCl and dil. to 100 mL. Soln is stable >1 
month. (2) Intermediate soln. — 50 jxg/mL. Dil. 10 mL stock 
soln to 100 mL with 0. 17V HCl. Soln is stable >1 month. (3) 
Working soln. — 5 juig/mL. Dil. 20.0 mL intermediate soln to 
200 mL with 0. 1W HCl. (Use same 0AN HCl as in extn of 
feed.) 

C. Determination 

Grind ca 100 g well mixed sample to pass No. 30 sieve and 
mix. (3 min in high-speed blender should be enough.) 

Weigh 2.000 g ground sample into 250 mL I 24/40 flat- 
bottom extn flask. (For feeds contg <0.025% thiabendazole, 
weigh 5.000 g.) 



Add 100.0 mL 0.1N HCl to sample and add mag. stirring 
bar. Connect flask to reflux condenser (Allihn, drip tip) and 
reflux gently on mag. hot plate, while stirring, 30 min. Cool, 
transfer mixt. to centrf. tube, and centrf. ca 5 min. Dil. mea- 
sured aliquot of supernate to concn of 5 fig thiabendazole/mL 
(serial dilns may be necessary). Such dilns det. u dilution fac- 
tor," DF: 



Declaration, 

% 



Sample 
wt, g 



Dtlution(s) 



DF 



0.01 


5 


none 


1 


0.025 


2 


none 


1 


0.1 


2 


25-100 


4 


1.0 


2 


10-100; 25-100 


40 


6,0 


2 


10-100; 10-250 


250 



Mark series of 50 mL centrf. tubes 1 , 2, 3, 4, etc. Place 
20.0 mL 0AN HCl in tube 1 and 20.0 mL (100 u.g) working 
std soln in tubes 2 and 3. Place 20.0 mL aliquots of sample 
solns in tubes 4, 5, etc. Add 3 g Na citrate, 3 g NaCl, and 
20.0 mL CHC1 3 to each tube, stopper tightly with polyethylene 
stopper, and shake mech. 5 min. Centrf. ca 5 min and discard 
top layers. With pipet, transfer 10 mL CHC1 ? ext to dry, marked, 
centrf. tubes, add 25.0 mL 0. IN HCl to each, stopper, and 
shake 5 min. Centrf., and transfer, with pipet, 15 mL of top 
acid layer to another marked tube. (Because of timing, handle 
<10 tubes at one time.) 

With rapid delivery pipet, add 5 mL freshly prepd Zn slurry, 
(b), to each tube. (5 mL pipet with tip cut off to give delivery 
in ca 5 sec is suitable. Hold pipet directly over center of soln.) 
Do not shake tube but immediately stopper tightly and let stand 
4 min. Start timing after delivery of slurry to first tube. 

After 4 min, add 5.0 mL ferric soln, (c), to each tube with 
rapid pipet, stopper, and mix by inverting tube. Let stand 5 
min; then shake vigorously and centrf. ca 3 min. With pipet, 
transfer 15 mL clear, colored soln to marked, dry, centrf. tubes. 
Let stand 45 min from addn of ferric soln. Then add 5.00 mL 
ai-BuOH and 3 g anhyd. Na 2 S0 4 to each tube. Stopper, and 
immediately shake each tube ca 5 sec to avoid caking of Na 2 S0 4 ; 
then shake all tubes ca 3 min or until Na 2 S0 4 is completely 
dissolved, and centrf. 

Transfer clear BuOH soln (top layer) to dry 1 cm cell and 
read A at 605 nm against n-BuOH as ref. 

% Thiabendazole in feed 

= (A - A )(C)(DF)/360(A f - A )W 

where A refers to sample, A to reagent blank (tube 1), A' to 
std, C = |ig thiabendazole std in final 15.0 mL colored soln 
(18 |xg), DF = diln factor, and W = g original sample. 

Refs.: JAOAC 47, 235(1964); 49, 312(1966). 

CAS- 148-79-8 (thiabendazole) 



966.29 Thiabendazole in Feeds, Supplements, 
and Premixes 
Spectrophotometric Method 

First Action 1966 
Final Action 1967 

Method II 

(Applicable to cattle supplements and premixes contg >1% 
thiabendazole. Principle is same as 966. 28A, except that sin- 
gle extn at room temp, with 0.1/V HCl is used. Not applicable 
to feeds, premixes, or cattle supplements contg high levels of 
protein.) 



AOAC Official Methods of Analysis (1990) 



Antibiotics 



115 



A. Reagents 

See 966.28B except: 

(a) Thiabendazole working soln. — 2 |xg/mL. Dil. 10.0 mL 
thiabendazole intermediate soln, 966.28B(d)(2), to 250 mL with 
O.IjVHCI. 

B. Determination 

Prep, sample as in 966*28C, except use ca 50 g represen- 
tative sample. 

Weigh 2.000 g ground sample into 1 L vol. flask and add 
750 mL 0.17V HC1. Add mag. stirring bar, stopper, and mix 
vigorously on mag. stirrer 1 hr at room temp. (Mech. shaker 
providing vigorous agitation may be used.) Remove and rinse 
bar, and dil. to vol. with 0.1JV HCL Mix, centrf., and dil. 
aliquots of clear ext with 0. IN HC1 to concn of 2 jxg thiaben- 
dazole /mL. Diln factors, DF, are as follows (see 966. 28C): 



Declaration, 



Dilution 



DF 



1.0 
2.5 
6.0 



10-100 
4.0-100 
4.0-250 



10 
25 
62.5 



Develop color in exts as soon as possible after extn. (Acid 
exts of some feeds deteriorate upon standing.) 

Mark series of 50 mL centrf. tubes (<10) as in 966. 28C. 
Add 15.0 mL 0. \N HC1 to tube 1 , and 15.0 mL working soln, 
(a), to tubes 2 and 3. Add 15.0 mL sample exts to other tubes. 
Then with rapid delivery pipet, add 5.0 mL freshly prepd Zn 
slurry as in 966. 28C. Proceed as in 966. 28C with addn of 
ferric soln, observing same technics and time precautions. Read 
final clear BuOH ext, as above, in 1 cm cell at 605 nm. 

% Thiabendazole in feed 

- (A - A )(C)(DF)/90(A' - A )W 

where symbols are as defined in 966. 28C. 

Rets.: JAOAC 47, 235(1964); 49, 312(1966). 

CAS- 148-79-8 (thiabendazole) 



(f ) Zoalene std soln. — 40 |xg/m'L. Weigh 40.0 mg Zoalene 
Ref. Std (available from Dow Chemical Co.) into 1 L vol. 
flask, dil. to vol. with 85% CH 3 CN, and mix. 

C. Determination 

Weigh 10.0 g sample into 250 mL erlenmeyer and add 65 
mL 85% CH 3 CN. Warm on steam bath to 50 ± 5°, swirling 
occasionally. Let cool to room temp, (ca 30 min). Add 20 g 
alumina and swirl occasionally ca 3 min. (Addn of alumina is 
unnecessary for cones contg >1% zoalene.) Filter with suction 
on medium or fine porosity 40 mm diam. fritted glass funnel, 
transferring as much solids as possible. Transfer remaining solids 
with min. vol. 85% CH 3 CN, and suck dry. Suspend cake in 
funnel with min. vol. 85% CH 3 CN and slight stirring but with- 
out suction. Then filter with suction and repeat suspension and 
filtering, keeping total vol. <100 mL. Transfer combined fil- 
trates to 100 mL vol. flask (or vol. flask may be used to collect 
filtrates directly), dil. to vol. with 85% CH 3 CN, and mix. 

Based on zoalene concn, make addnl dilns with 95% ace- 
tone and use aliquots indicated in Table 961.26. 

Pipet indicated aliquots into three 50 mL beakers, X, Y, and 
Z, for conens <0.25%; omit X for samples >0.25%. Pipet 1 
mL std soln into beaker Z and evap. all solns to dryness with 
air current. (Heat may be used but temp, must not exceed 60°.) 
Pipet 10 mL 95% DMF into X and 2 mL each into Y and Z. 
Swirl intermittently during 5 min to dissolve zoalene. Pipet 8 
mL ethylenediamine into Y and Z and mix. If turbidity persists 
after 2 min, filter thru small Reeve Angel No. 804, or equiv., 
paper. Read A of solns at 560 nm in stoppered 1 cm cells 
against 95% DMF 5 min after addn of ethylenediamine. Keep 
cell compartment of spectrophtr at <30° to avoid rapid fading 
of color. If A is >1 , reanalyze, using greater diln or smaller 
aliquot. 



% Zoalene = (A Y - A x ) x M/100 (A z - A Y ) 



Caution: CH 3 CN and ethylenediamine are toxic. 
hood and avoid contact with skin. 



Handle in 



Refs.: JAOAC 44, 18(1961); 45, 294(1962); 51, 501(1968). 
CAS- 148-01-6 (zoalene) 



961.26 Zoalene in Feeds 

Spectrophotometric Method 

First Action 1961 
Final Action 1962 

(Not applicable in presence of furazolidone, nitrofurazone, and 
nihydrazone) 

A. Principle 

Zoalene is extd from feeds, premixes, and cones contg 0.004- 
25% with 85% CH 3 CN. For mixes contg <1%, alumina is 
added. After filtration and diln, zoalene is detd colorimetrical- 
ly after reaction with ethylenediamine. 

B. Reagents 

(a) Acetone. — 95%. Add 5 mL H 2 Q to 95 mL acetone. 

(b) Acetonitrile—%5%. Add 850 mL practical grade CH 3 CN 
to 150 mL H 2 (deionized or distd). 

(c) Activated alumina. — Alcoa grade F 20, 80-200 mesh. 
(Available from Fisher Scientific Co. "Alumina, Adsorption, 
Fisher. ") 

(d) Dimethylformamide (DMF).— 95%. Add 5 mL H 2 to 
95 mL tech. DMF. Prep, fresh daily, since old solns may cause 
cloudiness. (Caution: see safety notes on dimethylformamide.) 

(e) Ethylenediamine. —98- 100%. (No. EX0510, EM Sci- 
ence). Reagent must be practically colorless. 



Table 961.26 Dilution of Sample for Determination 



% Zoalene 
in Sample 



Addnl Diln 



Aliquot 
Size, mL 



Multiplication 
Factor M 



0.004- 0.012 


None 


4 


1 


0.012- 0.025 


None 


2 


2 


0.025- 0.050 


10 to 100 


10 


4 


0.050- 0.10 


10 to 100 


5 


8 


0.10 - 0.25 


10 to 100 


2 


20 


0.25 - 0.5 


1 to 100 


10 


40 


0.5 - 1.0 


1 to 100 


5 


80 


1.0 - 2.5 


1 to 100 


2 


200 


2.5 - 5.0 


1 to 1000 


10 


400 


5.0 -10.0 


1 to 1000 


5 


800 


10.0 -25.0 


1 to 1000 


2 


2000 



MICROBIOLOGICAL METHODS FOR ANTIBIOTICS 



957.23 



Antibiotics in Feeds 
Microbiological Methods 



A. Culture Media 

(Deionized H 2 may be used for prepn of media.) 

(a) Agar medium A. — (Antibiotic Medium 1 .) Dissolve 6.0 
g pancreatic digest of gelatin, 4.0 g pancreatic digest of casein, 



116 



Drugs in Feeds 



AOAC Official Methods of Analysis (1990) 



3.0 g yeast ext, 1.5 g beef ext, 1.0 g anhyd. glucose, and 15 
g agar in H 2 0, and dil. to 1 L. Adjust with IN NaOH or HC1 
(1 + 9) so that after sterilization pH is 6.5-6.6. (Difco Pen- 
assay Seed Agar (antibiotic medium I) and BBL Seed Agar 
have been found satisfactory.) 

(b) Agar medium B. — (Antibiotic Medium 4.) Dissolve 6.0 
g pancreatic digest of gelatin, 3.0 g yeast ext, 1.5 g beef ext, 
1.0 g anhyd. glucose, and 15 g agar in H 2 0, and dil. to 1 L. 
Adjust with IN NaOH or HC1 (1 + 9) so that after sterilization 
pH is 6.5-6.6. (Difco and BBL Yeast Beef Agar have been 
found satisfactory.) 

(c) Agar medium C. — (Antibiotic Medium 2.) Dissolve 6.0 
g pancreatic digest of gelatin, 3.0 g yeast ext, 1,5 g beef ext, 
and 15 g agar in H 2 0, and dil. to 1 L. Adjust with IN NaOH 
or HC1 (1 +9) so that after sterilization pH is 6.5-6.6. (Difco 
Penassay Base Agar and BBL Base Agar have been found sat- 
isfactory.) 

(d) Agar medium D. — (Antibiotic Medium 8.) Use agar 
medium C adjusted with 17V NaOH or HC1 (1+9) so that 
final pH is 5.7-5.9. (Difco Antibiotic Medium 8 and BBL 
Base Agar with low pH have been found satisfactory.) 

(e) Agar medium E. — (Antibiotic Medium 5.) Use agar me- 
dium C adjusted with IN NaOH so that final pH is 7.8-8.0. 
(Difco Streptomycin Assay Agar (antibiotic medium 5) and 
BBL Streptomycin Assay Agar with Yeast Extract have been 
found satisfactory.) 

(f) Agar medium, F. — Adjust agar medium A with 3.57V 
NaOH (2.8-3.8 mL/L) so that after sterilization pH is 8.9- 
9.1. 

(g) Agar medium G. — (Antibiotic Medium 32.) Use agar 
medium A to which is added 300 mg MnS0 4 .H 2 or 0.4 ml 
.1% MnCl 2 soln/L. 

(h) Agar medium H. — Dil. 1 L agar medium A to 1.2 L 
and adjust to pH 8.1. 

(i) Agar medium. I. — Dissolve 9.4 g pancreatic digest of 
gelatin, 4.7 g yeast ext, 2.4 g beef ext, 10. g NaCl, 10.0 g 
anhyd. glucose, 13.0 g anhyd. KH 2 P0 4 , 1 g Na 2 HP0 4 .7H 2 0, 
and 23.5 g agar in H 2 0, and dil. to 1 L. After sterilization pH 
is 5.3-5.5. (BBL Nystatin Assay Agar supplemented with 13.0 
g anhyd. KH 2 P0 4 and 1 g Na 2 HP0 4 .7H 2 has been found sat- 
isfactory.) 

(j) Agar medium J. — (Antibiotic Medium 11.) Use agar 
medium A adjusted with IN NaOH so that final pH is 7.9- 
8.0. (Difco and BBL Neomycin Assay Agar have been found 
satisfactory.) 

(k) Agar medium K. — To each L agar medium J add 12.5 
mL 2M CaCl 2 after autoclaving and just before pouring plates. 

(J) Agar medium L.— Dissolve 0.69 g K 2 HP0 4 , 0.45 g 
KH 2 P0 4 , 2.5 g yeast ext, 10.0 g anhyd. glucose, and 15.0 g 
Difco Noble agar in H 2 and dil. to 1 L. Adjust to pH 6.0 
with HC1 before use. 

(m) Agar medium M. —Dissolve 2.5 g yeast ext, 10.0 g 
glucose, 0.69 g K 2 HP0 4 , 0.45 g KH 2 P0 4 , and 20.0 g agar in 
H 2 and dil. to 1 L. Before adding inoculum, adjust liquified 
medium to pH 6.0 with IN HC1 (ca 2 mL/L). 

(n) Broth medium A. — (Antibiotic Medium 3.) Dissolve 5.0 
g pancreatic digest of gelatin, 1.5 g yeast ext, 1 .5 g beef ext, 
3.5 g NaCl, 1.0 g anhyd. glucose, 3.68 g anhyd. K 2 HP0 4 , 
and 1.32 g anhyd. KH 2 P0 4 in H 2 0, and dil. to 1 L. Adjust 
with IN NaOH or HCJ (1 +9) so that after sterilization pH 
is 6.95-7.05. (Difco Penassay Broth (antibiotic medium 3) and 
BBL Antibiotic Assay Broth have been found satisfactory.) 

(o) Broth medium B. — Dissolve 5.0 g pancreatic digest of 
casein, 5.0 g pancreatic digest of animal tissues, and 20 g 
anhyd. glucose in H 2 0, and dil. to 1 L. Adjust with IN NaOH 
or with HC1 (1 +11) so that after sterilization pH is 5.6-5.7. 
(Difco Fluid Sabouraud Medium and BBL Sabouraud Liquid 
Broth Modified have been found satisfactory.) 



B. Reagents 

(a) Phosphate-bicarbonate buffer. — pH 8. Dissolve 16.73 
g anhyd. K 2 HP0 4 , 0.523 g anhyd. KH 2 P0 4 , and 20 g NaHC0 3 
in H 2 and dil. to 1 L. 

(b) Phosphate buffer.— pH 8; 0.1M. Dissolve 16.73 g 
anhyd. K 2 HP0 4 and 0.523 g anhyd. KH 2 P0 4 in H 2 and dil. 
to 1 L. 

(c) Phosphate buffer. — pH 7.0; 0.IM. Dissolve 13.6 g 
anhyd. K 2 HP0 4 and 4.0 g anhyd. KH 2 P0 4 in H 2 and dil. to 
1 L. 

(d) 5% Phosphate buffer.— pH 6.5. Dissolve 22.15 g 
anhyd. K 2 HP0 4 and 27.85 g anhyd. KH 2 P0 4 in H 2 and dil. 
to 1 L. 

(e) 10% Phosphate buffer. — pH 6. Dissolve 80 g anhyd. 
KH 2 P0 4 and 20 g anhyd. K 2 HP0 4 in H 2 and dil. to 1 L. 

(f) 1% Phosphate buffer. — pH 6. Dissolve 8.0 g anhyd. 
KH 2 P0 4 and 2.0 g anhyd. K 2 HP0 4 in H 2 and dil. to J L. 

(g) Phosphate buffer. — pH 4.5; 0.1 A/. Dissolve 13.6 g 
anhyd. KH 2 P0 4 in H 2 and dil. to 1 L. 

(h) Acid-acetone. — Mix 1 vol. AN HO, 13 vols acetone, 
and 6 vols H 2 0. 

(i) Acid-methanol. — Mix 1 vol. HC1 and 50 vols MeOH. 

(j) Ethyl acetate. — 99% undenatured grade. 

(k) Buffer-acetone extractant. — Mix equal vols pH 6 buffer, 
(f), and acetone. 

(1) Tris buffer.— pH 8.0, 0.05M. Dissolve 6.05 g 
tris(hydroxymethyl)aminomethane (THAM, primary std, 
available from Fisher Scientific Co.) in 900 mL H 2 0, adjust 
pH to 8.0 with HC1, and dil. to 1 L. 

(m) Calcium chloride soln. — 2M. Dissolve 294.04 g 
CaCl 2 .2H 2 in H 2 and dil. to I L. 

(n) Sodium chloride-calcium chloride soln. — Dissolve 200 
g NaCL in H 2 0, add 10 mL 2M CaCl 2 , and dil. to 1 L. 

(o) Sodium hypochlorite soln. — 5.25%. Use freshly opened 
bottle com. soln. (Clorox has been found satisfactory.) Store 
in dark at 2-10°. 

(p) Sterile isotonic saline soln. — Dissolve 9.0 g NaCl in 
H 2 and dil. to I L. Autoclave 20 min at 121°. 

(q) Lead acetate soln. — Dissolve 303 mg Pb(OAc) 2 .3H 2 
in H 2 and dil. to 1 L with H 2 0. 

C. Apparatus 

(High-speed blender jars, after disassembling, must be cleaned 

with great care to eliminate all traces of antibiotics. All app. 

that contacts sample and solns must be thoroly cleaned and be 

detergent-free.) 

(a) Cylinders. — Polished open stainless steel cylinders, 8 ± 
0.1 mm od, 6 ± 0. 1 mm id, and 10 ± 0. 1 mm high (obtainable 
from S & L Metal Products Corp., 58-29 57th Drive, Mas- 
peth, NY 11378). 

(b) Petri dishes {plates). — Glass or plastic; 100 mm wide 
X 20 mm deep. Porcelain covers glazed on outside or cover 
lids with filter pad inserts are satisfactory for absorbing H 2 
of syneresis. Glass or plastic covers may be used if they are 
raised slightly to allow escape of H 2 0. 

(c) Cylinder dispenser. — May be used to place cylinders on 
plates. Shaw Dispenser, available from Arthur E. Farmer, 47 
Frazier St, PO Box 1785, Trenton, NJ 08618. 

(d) Agar cutter — Used to prep, cups in agar, available from 
Biochem. Dept, Purdue Univ., W. Lafayette, IN 47907. 

D. Stock Cultures and Preparation of Test Organism 
Suspensions 

For appropriate test organism designated below, prep, slant 
culture on >1 tube of agar medium A. Incubate overnight at 
indicated temp, held constant to ± 0.5°, and then store in dark 
at 2-10°. Do not use if >2 weeks old. 



AOAC Official Methods of Analysis (1990) 



Antibiotics 



117 



Prep, suspensions of test organisms as follows: 

(a) Micrococcus flavus . — ATCC No. 10240. Incubate stock 
culture at 32-35°. Wash growth from stock culture with ca 3 
m'L broth medium A and transfer liq. to surface of 300 mL 
agar medium A in Roux bottle. Spread suspension evenly over 
entire surface, using sterile glass beads, and incubate over- 
night at 32-35°. Wash growth from agar surface with ca 25 
mL sterile isotonic saline soln. Store bulk suspension at 2- 
10°. Use for bacitracin assay. 

(b) Sarcina subflava. — ATCC No. 7468. Incubate stock 
culture at 32-35°. Prep, suspension as in (a) and use as al- 
ternative organism for bacitracin assay. 

(c) Bacillus cereus. — ATCC No. 11778. Incubate stock 
culture at 30°. Wash growth from stock culture with ca 3 mL 
sterile H 2 0, transfer to surface of 300 mL agar medium A, 
and incubate 7 days at 30°. Wash growth from agar surface 
with ca 25 mL sterile H 2 and heat suspension 30 min at 65°. 
Centrf. and decant. Wash residual spores 3 times with sterile 
H 2 0, centrfg and decanting each time. Discard wash H 2 0. Heat 
residual spores 30 min at 65° and resuspend in sterile H 2 0. 
Store this stock suspension at 2-10°. Use for chlortetracycline 
and oxytetracycline assays. 

(d) Bacillus sub tills. — ATCC No. 6633. Incubate stock 
culture at 37°. Wash growth from stock culture with ca 3 mL 
sterile isotonic saline soln, transfer to surface of 300 mL agar 
medium G in Roux bottle, and incubate 7 days at 37°. Wash 
growth from agar surface with ca 50 mL sterile isotonic saline 
soln into centrf. bottle. Heat suspension 30 min in 65° H 2 
bath to destroy vegetative cells. Centrf., decant, and resuspend 
cells in ca 50 mL sterile isotonic saline soln. Repeat heating, 
centrfg, and suspending twice, or until supernate is clear. Final 
suspension is stock spore suspension. Store at 2-10°. Use for 
hygromycin B, monensin, and streptomycin assays. 

(e) Sarcina lutea. — ATCC No. 9341. Incubate stock cul- 
ture at 26-30°. Prep, organism suspension by one of following 
methods: 

(/) Roux bottle culture. — Wash growth from 24 hr slant 
culture with ca 3 mL broth medium A, and transfer liq. to 
surface of 300 mL agar medium A in Roux bottle. Spread 
suspension evenly over entire surface, using sterile glass beads, 
and incubate 24 hr at 26-32°. Wash growth from agar surface 
with ca 15 mL sterile isotonic saline soln. Store bulk suspen- 
sion <2 weeks at 2-10°. 

(2) Broth culture. — Wash growth from stock culture with 
ca 3 mL broth medium A, and transfer liq. to 100 mL broth 
medium A. Incubate 48 hr at 26-32° with continuous mech. 
agitation. This 48 hr culture is inoculum. Store <2 weeks at 
2-10°. 

Use for erythromycin, lincomycin, novobiocin feed supple- 
ment, oleandomycin, penicillin, and tylosin assays. 

(f) Staphylococcus epidermidis.^ ATCC 12228. Incubate 
stock culture at 32°. Inoculate 30 mL broth medium A in 300 
mL flask with 1 loop from stock culture, and incubate over- 
night at 26-32°. Prep, daily. Use for neomycin and for no- 
vobiocin final feed assays. 

(g) Saccharomyces cerevisiae. — ATCC No. 9763. Incu- 
bate stock culture on agar medium I at 37°. Prep, inoculum 
by one of following methods: 

(/) Broth culture. — Inoculate 100 mL broth medium B with 
1 loop from stock culture and incubate overnight at 37°. This 
culture is inoculum. Store <2 weeks at 2-10°. 

(2) Roux bottle culture. — Wash growth from stock culture 
with ca 3 mL sterile isotonic saline soln, and transfer liq. to 
surface of 300 mL agar medium I in Roux bottle. Spread sus- 
pension evenly over entire surface, using sterile glass beads, 
and incubate 24 hr at 37°. Wash growth from agar surface with 
ca 15 mL sterile isotonic saline soln. Store <2 weeks at 2- 
10°. 



Use for nystatin assay. 

(h) Escherichia cob. — UC 527 (available from The Upjohn 
Co.). Incubate at 36°. Inoculate 30 mL broth medium A in 
250 mL flask from stock culture of E. coli and grow 1.8-24 
hr at 36°. Prep, daily. Use for spectinomycin assay. 

£. Design and Plotting of Standard Response Line 

Prep, concns of ref. std as described for each antibiotic. In 
general, it is preferable to use shorter 4-fold range between 
lowest and highest doses of std line. Use indicated concn as 
ref. concn. (Values of std or ref. concn could slightly vary 
from those indicated for each antibiotic without affecting va- 
lidity of assay.) 

Prep, plates with appropriate base agar layer and /or appro- 
priate seed agar layer; one layer of media can be substituted 
for 2 layers of media if ref. concn gives adequate zone size 
as described for each antibiotic. Distribute agar evenly by tilt- 
ing plates from side to side with circular motion and let harden. 
Use plates same day prepd. 

Place 6 cylinders on each plate at ca 60° intervals on 2.8 
cm radius. Fill 3 alternate cylinders with ref. concn and other 
3 cylinders with one of other concns of std. Use 3 plates for 
each concn required for std response line, except ref. concn. 
Incubate plates overnight at appropriate temp., and measure 
diams of zones of inhibition as accurately as possible. (In most 
cases, it is possible to est. zone diams to nearest 0.1 mm.) 
Values given in each method for zones of inhibition to be ob- 
tained with ref. concns of antibiotics are for guidance only, 
but it is important that lowest concns on std response line give 
measurable zone and that slope of response line be adequate. 
In each set of 3 plates, average the 9 readings of ref. concn 
and the 9 readings of concn being tested. Av. of all 36 read- 
ings of ref. concn from 12 plates is correction point for re- 
sponse line. Correct av. value obtained for each concn to ap- 
propriate figure if ref. concn reading on that set of 3 plates 
was same as correction point. 

For example, if in correcting second concn of std response 
line, av. of 36 readings of ref. concn is 20.0 mm, and av. of 
9 readings of ref. concn of this set of 3 plates is 19.8 mm, 
correction is + 0.2 mm. If av. reading of second concn on 
same 3 plates is 17.0 mm, corrected value is 17.2 mm. Plot 
corrected values, including correction point, on semilog graph 
paper, using logarithmic scale for concn and arithmetic scale 
for av. zone diams. Manual plotting of std lines is possible 
but could be subject to large variation. Response lines would 
be more accurate if calcd. When std doses are equally spaced, 
i.e., interval between successive doses is the same, calc. L 
and H (calcd zone diams for low and high concns, resp., of 
std response line) as follows: 

For method specifying 5 doses of std, 

L - (3a + 2b + c - e)/5 

H = (3e + 2d + c - a)/5 

where a, b, c, d, and e — corrected av. zone diams for each 
concn of std. 

For methods specifying 4 doses of std, 

L = (la + 4b 4- c - 2d)/\0 

H = (Id + Ac + b - 2a)/ 10 

For methods specifying 3 doses of std, 

L - (5a + 2b - c)/6 

H - (5c + 2b ~~ a)/6 

Plot values for L and H and connect with straight line. Ref. 
point is zone size intercept on arithmetic scale. This corrected 
ref. point is to be used for sample calcns (if corrected ref. point 
diam. varies significantly from av. ref. diam., error in prepn 



118 



Drugs in Feeds 



AOAC Official Methods of Analysis (1990) 



of std solns is indicated and validity of assay is in question.) 
For more accuracy in calcn, det. slope of std response line B 
= (H - L)/(log h — log /), where / and h are high and low 
std concns, resp., and B is increase in zone for each 10X in- 
crease in drug concn. 

Computer or calculator can be used to calc. std lines whether 
std concn s are equally spaced or not. Least square fitting using 
linear or polynomial equations may be performed based on 
best fit (polynomial fitting is most appropriate, especially for 
long range 8x or 16x range). 

F. Determination of Potency 

Use 3 plates of each assay soln. On each plate, fill 3 alter- 
nate cylinders with ref. concn and fill other 3 alternate cyl- 
inders with assay soln. Incubate plates overnight at appropriate 
temp, and measure diam. of zones of inhibition. Average the 
9 readings of ref. concn and the 9 readings of assay soln. If 
assay soln gives larger av. than ref. concn, add difference be- 
tween them to ref. point on std response line. If assay soln 
gives smaller value than ref. concn, subtract difference be- 
tween them from ref. point on std response line. Using cor- 
rected values of assay soln, det. amt of antibiotic by reading 
concn from std response line. 

Alternatively, det. log relative potency, M' = (Y u - Y s )/B, 
where Y u and K s are av. of 9 readings of assay soln and ref. 
concn, resp., and B is slope of std response line. Antilog M' 
= potency of assay soln relative to std; and (antilog M') x 
100 — potency of assay soln as % of std ref. concn. 

For calcn of sample potency by computer or calculator, en- 
ter sample data and calc. antibiotic potency based on least square 
linear or polynomial lines. 

For calcns, 1 ton = 908 000 g; 1 lb = 454 g. 

Refs.: JAOAC 40, 857(1957); 72, 105(1989). 



982.43 Bacitracin in Premix Feeds 

Cylinder Plate Method 
First Action 1982 

(Applicable to premixes contg ^10 g bacitracin/lb) 

A. Principle 

Bacitracin is extd from feeds into acidified org. solv. sys- 
tem. Ext is centrfd, and supernate is dild in phosphate buffer 
and analyzed by cylinder plate assay with M. flavus as detec- 
tion organism. 

B. Reagents and Apparatus 

(a) Microorganism. — Micrococcus flavus ATCC 10240. 
Maintain culture as indicated in 957.23D(a). 

(b) Extracting solv.— -Mix, by vol., 27% CH 3 CN, 27% 
MeOH, 3% pH 6.0 phosphate buffer (957.236(f)), 41% H 2 0, 
and 2% H 3 P0 4 (85%); add 0.5 g EDTA/L. (Extg solv. is satd 
with EDTA.). 

(c) Phosphate buffer. -—5%, pH 6.5. See 957.238(d). 

(d) Diluting solvent.—- Methanol -5% pH 6.5 phosphate 
buffer (12 + 88). 

(e) Dilute HCL— Carefully add 89 mL HC1 to H 2 and dil. 
to 1 L (IN). Further dil. soln 1:100 (0.01N). 

(f) Cylinders. —See 957.23C(a). 

(g) Cylinder dispenser. — Optional: see 957.23C(c). 

C. Standard Solutions 

See 957.24*(a) and (b). Also prep. 0.30 and 0.16 unit/mL 
solns to be plated as samples to monitor assay. 



D. Preparation of Plates 

Use one layer (ca 15 mL) of agar antibiotic medium, 1, 
957.23A(a). Det. by trial plates optimum concn (usually 0.02- 
0.05%) of M. flavus ATCC 10240 to be added to agar to ob- 
tain zones of inhibition 15-17 mm for 0.2 unit bacitracin/mL. 
Pour 4 plates for each point on std curve (i.e., 16 plates) and 
4 plates for each sample soln. Std curve will be plated twice 
(i.e., 32 plates) as will check samples 0.30 and 0.16 unit/mL. 
Therefore, total of 48 plates will be needed for 2 curves and 
check samples, plus 4 addnl plates for each sample. 

Let agar harden on level surface. Transfer to refrigerator and 
cool ^1 h before dosing. Use plates same day prepd. 

£ Extraction 

Accurately weigh amt feed contg ca 4600 units of bacitracin 
into 300 mL erlenmeyer flask, or equiv. 

Add 100 mL extg solv. with 100 mL vol. pipet and ext feeds 
5=5 min by shaking flask or mixing on mag. stirrer. 

Transfer supernate to plastic centrif. tubes and centrif. 10 
min at 2000 rpm. Filter supernate thru glass wool into grad- 
uate. Use vol. glassware and dilg solv. to prep, final diln 0.2 
± 0.05 unit/mL. 

F. Plating 

Use 16 seeded plates for first curve. Use 0.20 unit/mL as 
plate ref. On each plate, fill 3 alternate stainless steel cylinders 
with plate ref. and the 3 remaining cylinders with 1 std. Be 
sure all cylinders are filled with const vol. (i.e., 0.25 mL). 
Preset Eppendorf pipet is best for this purpose. Use 4 plates 
for each sample, including 0.3 and 0.16 unit/mL check sam- 
ples. 

Use 16 seeded plates for second curve, to be plated after all 
samples are plated. Use 8 plates for second plating of 0.3 and 
0.16 unit/mL check samples. 

incubate dosed plates 16-18 h at 37 ± 2°. Read zones of 
inhibition to nearest mm, using Fisher-Lily zone reader. 

G. Determination 

Det. corrected av. zone diams for std (Z') and sample (Z) 
solns according to 957. 23E. Det. response line as least squares 
linear regression of following equation: 

Z' = mlog P' + b 

where P' = potency in unit/mL of std soln associated with 
Z'; m, b = are least squares fitted slope and intercept param- 
eters. Calc. potency of sample by following equation: 

g bacitracin/lb = [antilog(Z - b/m) 

x D x 0.0108]/sample wt 

where D = total sample diln; 0.0108 - 453.6 (g/lb)/42 000 
(units/g bacitracin). 

Ref.: JAOAC 65, 1168(1982). 

CAS-1405-87-4 (bacitracin) 



957,24* Bacitracin 

in Feed Supplements 
Microbiological Method 

First Action 1957 

Final Action 1960 

Surplus 1981 

(Applicable to supplements contg 5=6 g/lb) 
See 42.223-42.226, 14th ed. 



AOAC Official Methods of Analysis (1990) 



Chlortetracycline 



119 



965.48* Bacitracin in Mixed Feeds 

Microbiological Method 

First Action 1965 
Surplus 1981 

(Applicable to feeds contg ^20 g bacitracin /ton) 
See 42.227-42.231, 14th ed. 



967.39 Chlortetracycline HCI in Feeds 

Microbiological Method 
First Action 1967 
Final Action 1968 

(Applicable to feeds contg >:10 ppm) 

A. Standard Solutions 

(a) Chlortetracycline (CTC) stock soln. — Accurately weigh 
ca 40 mg CTC. HCI USP Ref. Std and dissolve in enough 0.0 IN 
HCI to give concn of exactly 1000 jxg/mL. Store in dark <5 
days at 2-10°. 

(b) Std solns and response line for samples containing more 
than 50 ppm chlortetracycline. HCI. — Dil. appropriate ali- 
quots of stock soln, (a), with enough pH 4.5 buffer, 957.23B(g), 
to obtain conens of 0.01, 0.02, 0.04, 0.08, and 0.16 >xg/mL. 
Ref. concn is 0.04 |xg/mL. 

(c) Std solns and response line for samples containing 10— 
50 ppm chlortetracycline .HCI. — Prep, as in (b), but dil. with 
inactivated diluent, (d), instead of buffer soln and include conens 
of 0, 0.005, and 0.32 \ig CTC.HCl/mL. Draw best line of fit 
by inspection. 

(d) Inactivated diluent. — To 10 ml acid-acetone feed ext 
(prepd from feed under test as in 967.39C(b)) in 600 mL beaker, 
add 90 mL pH 4.5 phosphate buffer, 957.23B(g), and adjust 
to pH 4.5-4.7 with IN NaOH. Add 1.0 mL fresh 5.25% NaOCl 
soln, 957.23B(o), and stir 1-2 min, rinsing sides of beaker. 
Heat, stirring thoroly at 10 min intervals, in uncovered beaker 
in boiling H 2 bath 30 min. Cool to room temp, under tap 
H 2 stream and transfer quant, to 100 mL vol. flask. Rinse 
beaker with 6 mL acetone, add rinsings to vol. flask, and dil. 
to vol. with pH 4.5 buffer. Transfer quant, to another flask 
and dil. with enough pH 4.5 buffer so that final concn of feed 
ext is same as that in assay soln. 

B. Plates 

(a) Base layer. — Add 6.0 mL melted agar medium D to 
sterile petri dishes, distribute evenly, and let harden on per- 
fectly level surface. 

(b) Seed layer.— Before assay, det. by prepn of trial plates 
optimum concn (usually 0.03-0. 10%) of organism suspension 
of B. cereus, 957.23D(c), to be added to agar medium D to 
obtain zones of inhibition with as little as 0.01 |xg CTC. HCI/ 
mL for assaying samples contg >50 ppm and 0.005 |xg/mL 
for samples contg <50 ppm CTC. HCI. Zone of 20 mm ± 10% 
should be obtained with 0.04 |mg/mL. For actual assay add 
appropriate amt of suspension to agar medium D previously 
melted and cooled to 48°. Mix thoroly and add 4.0 mL to each 
of plates contg base layer. Alternatively, use 10-12 |xL agar 
medium D (single layer). 

C. Assay Solution 

(Solv. losses may occur from evapn of volatile solvs in open 
containers. Carefully measure and record vols of solvs, and 
make appropriate mathematical corrections for any losses in 

vol.) 



(a) Samples containing more than 50 ppm chlortetracy- 
cline. HCI. — Obtain and prep, sample as in 965.16 and 950.02. 
Place 2, 10, or 20 g sample, resp., contg CTC. HCI >10 g/lb 
(>2%), >400 ppm to 2%, or 50-400 ppm in 150 mL beaker 
and pipet in 40 mL acid-acetone soln, 957.23B(h). Stir ca 2 
min with glass rod, let stand 2 min, and stir. Adjust pH to 
1.0-1.2 with HCI, if necessary, and note vol. HCI added. 
Transfer to 1 qt (1 L) high-speed blender jar, using addni 20 
mL (minus vol. equiv. to HCI added in adjusting pH) acid- 
acetone to rinse beaker and pH meter electrodes. Cover jar and 
blend 3 min at high speed. Transfer mixt. to 100 mL centrf. 
tubes. Wash blender jar with 40 mL acid-acetone and combine 
washings with ext in centrf. tubes. Shake well 5 min. Centrf. 
ca 15 min at 2000 rpm. Combine and mix clarified exts. Ad- 
just 10 mL aliquot to pH 4.5 with IN NaOH. Dil. adjusted 
soln with enough pH 4.5 buffer, 957.23B(g), to obtain estd 
concn of 0.04 jjig/mL. Designate soln as assay soln. 

(b) Samples containing 10-50 ppm chlortetracycline. 
HCI. — Obtain and prep, sample as in 956.16 and 950.02. Place 
50 g sample in 250 mL beaker and pipet in 100 mL acid- 
acetone soln, 957.236(h). Stir, adjust pH, and blend as in (a), 
using 50 mL (less vol. equiv. to HCI added in adjusting pH) 
acid-acetone to transfer to blender jar. After blending, transfer 
quant, to 250 mL centrf. bottle, rinsing jar with 50 mL acid- 
acetone soln. Shake thoroly and centrf. ca 15 min at 2000 rpm. 
Pipet 5 mL clear supernate into 50 mL beaker, add ca 40 mL 
pH 4.5 buffer, 957.23B(g), mix, and adjust pH to 4.5-4.7 
with IN NaOH. Transfer quant, to flask, rinse beaker and pH 
meter electrodes with pH 4.5 buffer, and add rinsings to flask. 
Add enough pH 4.5 buffer to obtain estd concn of 0.04 [xg/ 
mL. Designate as assay soln. 

D. Assay 

Using CTC. HCI std response line, assay soln, and plates, 
proceed as in 957.23E-F, incubating at 30°. 

Refs.: JAOAC 40, 857(1957); 50, 446(1967). 

CAS-64-72-2 (chlortetracycline. HCI) 



977.37 Chlortetracycline HCI in Feeds 

Turbidi metric Method 
First Action 1977 

(Applicable to feed supplements contg ^20 g/lb) 

A. Apparatus and Reagents 

(a) Assay broth. — Prep, as in 957.23A(n), but in 1.7x 
quantity. 

(b) Homogenizer. — Omni-Mixer (Du Pont Instrument Co., 
Sorvall Operations, Peck's Ln, Newtown, CT 06470), or equiv. 

(c) For manual assay . — (7 ) Spectrophotometer . — Sequoia- 
Turner Model 330 (replacement Model 340, Sequoia-Turner 
Corp., 850 Maude Ave, Mountain View, CA 94043), or equiv. 
Response time must be rapid, <4 sec. (2) Flowcell. — 10 mm 
light path and 0.25 mL vol. (No. 8495-L10, Thomas Scien- 
tific, or equiv.) and adapter (No. 8475-F10, Thomas Scien- 
tific, or equiv.) to hold cell assembly in spectrophtr. Polyeth- 
ylene tubing, 0.055" (1 .4 mm) id, is used as inlet and outlet. 
Fit inlet tube with short length of stainless steel tube and con- 
nect outlet to vac. thru solenoid valve (AU-0034, Elanco Prod- 
ucts Co., or equiv.). Adjust vac. to obtain flow rate of 1.0 
mL/sec. Flowcell must be rigidly held in its holder and holder 
rigidly fixed in adapter. (3) Water bath and heater. — Part of 
AUTOTURB System, (d), or equiv. (4) Constant voltage 
transformer. — Sola transformer, wave form corrected, for sta- 



120 



Drugs in Feeds 



AOAC Official Methods of Analysis (1B90) 



bilizing current voltage (EU-0020, Elanco Products Co., or 
equiv.); connected to spectrophtr. (5) Filling unit. — Filamatic 
single nozzle liquid filler (National Instrument Co., Inc., 41 19 
Fordleigh Rd, Baltimore, MD 21215), or equiv. Use with Te- 
flon tubing, 0.063" (1.6 mm) id, to fill assay tubes. (6) Digital 
voltmeter. — 3 l / 2 or 4 l / 2 digit. Newport Model 400AS3 (New- 
port Electronics, Inc., 630 E Young St, Santa Ana, CA 92705), 
or equiv. Connect to spectrophtr output to measure % T. 

(d) For automated assay. — Autoturb System (Elanco Prod- 
ucts Co.). 

B. Standard Solutions 

(a) For manual assay. — Dil. aliquots of stock soln, 
967.39A(a), in enough pH 4.5 buffer, 957.23B(g), to give 
concns of 0.02, 0.04, 0.06, 0.08, and 0.1.0 fxg CTC.HCl/mL. 

(b) For automated system. — Prep, concns of 0.2, 0.4, 0.6, 
and 0.8 p,g CTC.HCl/mL as in (a). 

C. Preparation of Inoculum 

Inoculate 200 mL broth medium A, 957.23A(n), with 1 loop 
from 24 hr stock culture of Staphylococcus aureus, ATCC 9144, 
and incubate overnight at 37° on rotary shaker. Store <2 weeks 
at 2-10°. 

D. Preparation of Samples 

Weigh 2 g sample into 250 mL glass or plastic centrf. bottle. 
Add 50 mL acid-acetone, 957.23B(h). Stopper or cap imme- 
diately, agitate intermittently 5 min, and adjust to pH 1.0-1.2 
with HC1, if necessary, using pH meter. Note vol. HC1 used. 
Add 50 mL (minus vol. HCJ used) of acid-acetone. Insert blades 
of homogenizer into centrf. bottle and blend 3 min at high 
speed while keeping bottle covered. Rinse blades into bottle 
with 100 mL acid-acetone. Tightly cap bottle and centrf. ca 
15 min at 2000 rpm. Filter thru Whatman No. 2V paper, or 
equiv. Make further dilns with pH 4.5 buffer, 957.23B(g), to 
ca 0.09 and 0.06 jxg CTC.HCl/mL for manual assay, and 0.6 
for automated assay. 

E. Assay 

(a) Manual method. — Inoculate assay broth, 957.23A(n), 
with 0.5-1 .0 mL inoculum, 977.37C/100 mL. Incubate at 37° 
(20-30 min) until A is ca 0.05 at 600 nm in 10 mm flowcell, 
using uninoculated broth as blank. 

Completely fill test tube carrier with 18 X 150 mm test tubes 
contg medium even tho assay may require only portion of these 
tubes. These tubes are included only to maintain uniform H 2 
flow in bath. 

Pipet 1 mL pH 4.5 buffer, 957.23B(g), into each of 4 blank 
tubes and into each of 4 zero level std tubes. Pipet into each 
of 4 tubes 1 mL of each std and each sample soln. Add 9.0 
mL inoculated assay broth to all tubes. Refrigerate blank tubes. 
Incubate all other tubes at 37° until % T of zero level tubes is 
ca 30 at 600 nin (3-4 hr). Do not remove tubes from bath 
during incubation to observe growth. Use extra tubes for this 
purpose and after inspection, replace in bath but do not mea- 
sure. Stop growth in all tubes by heating 1-2 min at 80°; then 
cool rapidly in cold H 2 0. Shake each tube by placing thumb 
over tube and inverting once. Do not shake mech. Measure 
turbidity at 600 nm in static suspension. Let culture flow ca 
4 sec, stop flow ca 2 sec to dislodge air bubbles, and let flow 
again ca 3 sec. Stop flow and read % T. Average the 4 read- 
ings for each std and sample. 

(b) Automated method. — System pipets two 0.10 and two 
0.15 mL portions of sample soln and std solns into assay tubes, 
dils with inoculated broth, and reads % T at 600 nm. Average 
the 2 readings. 



F. Calculations 

(a) Manual assay. — Convert av. % T to A and plot log A- 
against jxg CTC.HCl/mL on semi- log paper. Draw std re- 
sponse line. Read jxg CTC.HCl/mL in sample from line. 

(b) Automated assay . — Read |xg CTC.HCl/mL from graph 
made as in (a) for 0.10 mL vols and for 0.15 mL vols. Av- 
erage results. 

g/lb - jxg CTC.HC1 (from curve) x D x 454 

x 10~ 6 /g sample 

where D = diln factor, 454 X 1 0~ 6 - conversion of |xg/g to 
g/lb- 

Ref.: JAOAC60, 1119(1977). 

CAS-64-72-2 (chlortetracycline.HCl) 



971.48 Erythromycin in Feeds 

Microbiological Method 
First Action 1971 

(Applicable to feeds contg 9.25 and 92.5 g/ton without 

pelleting adjuvants and ^92.5 g/ton with bentonite or Ma- 

sonex) 

A. Reagent 

Dimethoxy methane {methylal). — Tech., CH 2 (OCH 3 ) 2 (Aid- 
rich Chemical Co., Inc.; or Eastman Kodak No. 525). 

B. Standard Solution 

(a) Erythromycin stock soln. — Accurately weigh amt USP 
Erythromycin Ref. Std and dissolve in enough methylal -MeOH 
(4 + 1) to give concn of 1000 (xg erythromycin base/mL. (1 
(xg base is equiv, to 1.08 |xg of the thiocyanate.) Dil. further 
with pH 8 buffer, 957.23B(b), to final concn of 100 p,g/mL. 
Store in refrigerator ^1 week. 

(b) Std response line. — Dil. appropriate aliquots of stock 
soln, (a), with enough pH 8 buffer, 957.23B(b), to obtain concns 
of 0.05, 0.1, 0.2, 0.4, and 0.8 u.g erythromycin base/mL. 
Ref. concn is 0.2 jxg/mL. 

C. Plates 

Before assay, det. by prepn of trial plates optimum concn 
(usually 0.05-0.2%) of organism suspension of Sarcina lutea, 
957.23D(e)(i), to be added to agar medium J, 957.23A(j), to 
obtain zones of inhibition of adequate size (17.5 mm ± 10% 
with ref. concn) and sharpness. For actual assay add appro- 
priate amt suspension to agar medium J previously melted and 
cooled to 48°. Place 10 mL inoculated medium in each of re- 
quired number of plates, let harden, and refrigerate in inverted 
position until just before use. 

D. Assay Solution 

Accurately weigh ca 10 g sample contg equiv. of >92.5 g 
erythromycin base /ton or 40 g contg equiv. of 9.25 g eryth- 
romycin base /ton and transfer to 250 mL g-s erlenmeyer. 

For feeds contg no pelleting adjuvants add 20.0 mL H 2 
and 80.0 mL methylal-MeOH (4 + 1) soln. For feeds contg 
bentonite or Masonex, add 20.0 mL 5% phosphate buffer, 
957.23B(b), and 15.0 mL MeOH. Mix and let feed slurry stand 
10 min before adding 65.0 mL methylal. Stopper and mix 1 
hr on mag. stirrer or mech. shaker (add glass beads for ade- 
quate mixing on shaker). 

Let feed settle and dil. to 0.203 p,g erythromycin base/mL 
as in (a) or (b) below: 



AOAC Official Methods of Analysis (1990) 



Hygromycin B 



121 



(a) Feeds containing equivalent of, or more than 92.5 g 
erythromycin base /ton. — Dil. 2.0 ml ext to 100 mL with pH 
8 buffer, 957.238(b). 

(b) Feeds containing equivalent of 925 g erythromycin base/ 
ton.— Dil. 5 mL ext to 100 mL with pH 8 buffer. 

E. Assay 

Using erythromycin std response line, 971.48B(b), assay soln, 
971.48D, and plates, 971. 48C, proceed as in 957.23E-F, ex- 
cept use 4 plates for each concn required for std response line 
(total of 16 plates) and for each assay soln. Incubate at 30°. 

Refs.: JAOAC 54, 940, 944(1971); 60, 176(1977). 
CAS- 11 4-07-8 (erythromycin) 



960.67 Hygromycin B in Feeds 

Microbiological Method 
Final Action 1965 

(Applicable to feeds contg >6000 units /lb) 

A. Standard Solutions 

(a) Hygromycin B stock soln. — Accurately weigh amt of 
Hygromycin B Ref. Std (available from Elanco Products Co.) 
contg 50,000 units, transfer to 50 mL vol. flask, and dil. to 
vol. with pH 7 phosphate buffer, 957.23B(c). Store in refrig- 
erator <2 weeks. 

(b) Std response line. — Dil. appropriate aliquots of stock 
soln daily with enough pH 7 buffer, 957.23B(c), to obtain 
concns of 15, 25, 50, and 75 units/mL. Ref. concn is 25 units/ 
mL. 

B. Plates 

(a) Base layer. — Add 10 mL melted agar medium E to ster- 
ile petri dishes, distribute evenly, and let harden on perfectly 
level surface. 

(b) Seed layer. — Before assay, det. by prepn of trial plates 
optimum concn (usually 0.2% of 1:10 diin) of spore suspen- 
sion of B. subtilis, 957.23D(d), to be added to agar medium 
E. Zone of 16 mm ± 10% should be obtained with 25 units/ 
mL. For actual assay add appropriate amt of spore suspension 
to agar medium E which has been melted and cooled to 48°. 
Mix thoroly and add 4.0 mL to each plate contg base layer. 
Store plates at 2-10° until just before use. 

C. Assay Solution 

(a) Preparation of ion exchange resin column. — Slurry ca 
1 lb (450 g) Amberlite IRC-50 ion exchange resin with 2 L 
IN H 2 S0 4 3 hr. Wash until neut. with H 2 and gradually add 
solid LiOH with stirring until pH remains at 7-8. Let stand 
overnight and wash with H 2 ^5 times. Neutze to pH 7.0 with 
IN H3PO4. Store under H 2 in glass container. 

Place glass wool plug at bottom of 6 mm id X 140 mm long 
tube fitted with valve to control flow and 50 mL reservoir at 
top. Fill tube with H 2 and add wet resin to within 20 mm of 
top of tube. Drain H 2 to within 5 mm of resin surface. Wash 
with 25 mL sterile H 2 immediately before use. 

(b) Preparation of assay soln. — Obtain and prep, sample 
as in 965.16 and 950.02. Weigh 50 g sample contg 6000- 
12,000 units/lb (30 g for 18,000-24,000 units/lb, 20 g for 
> 24, 000) into jar of high-speed blender. Add 300 mL (500 
for the higher potency feeds) pH 7 phosphate buffer, 957.23B(c), 
and blend 5 min, operating blender from variable transformer 
set at 70. Centrf. 10 min at 2600 rpm. Adjust 125 mL super- 
nate to pH 5.0 with HC1 (ca 0.5 mL). Add 50 mL CHC1 3 



previously washed with pH 7.0 buffer, stopper, and shake tho- 
roly. Centrf. mixt. 10 min at 2600 rpm. Remove aq. phase, 
adjust to pH 7.0 with 40% NaOH soln (ca 0.7 mL), and centrf. 
Transfer 100 mL neutzd soln (75 mL if feed contains > 42, 000 
units /lb) to ion exchange column and adjust flow rate to 40 
drops/min. Wash column with four 20 mL portions sterile H 2 0. 
Elute hygromycin B with 50 mL NH 4 OH (1 + 9) into 100 mL 
Pyrex beaker. Evap. to 3-5 mL and adjust to pH 7.0 with IN 
HC1. Transfer to 10 mL vol. flask, dil. to vol. with pH 7.0 
phosphate buffer, and designate as assay soln. (Final concn 
should be ca 25 units/mL.) 

D. Assay 

Using hygromycin std response line, assay soln, and plates, 
proceed as in 957.23E-F, except use 6 plates for each concn 
required for std response line (total of 18 plates) and for each 
assay soln. Equations forL and H cannot be used. Incubate at 
37°. 

E. Calculation 

Units/lb = [1.1 x (units/mL assay soln) x 454 x mL pH 
7 buffer (300 or 500) x 10 x (125 + mL HC1 + mL 40% 
NaOH)] /[ 125 mL x g sample x mL neutzd soln put on col- 
umn]. 

Ref.: JAOAC 43, 213(1960). 

CAS-3 1282-04-9 (hygromycin) 



975.60 



Lasalocid in Feeds 

Microbiological Method 

First Action 1975 



A. Reagents and Apparatus 

See 957.23C(a)-(c), 975.61C(a), and following: 

(a) Ethyl acetate. — Purify by passage over silica gel and 
distil. 

(b) Methanol.— 75% and 19.4% by vol. in H 2 0. 

(c) Automatic pipetting machine. — Brewer (available from 
Scientific Equipment Products (SEPCO), or equiv. 

B. Standard Solutions 

(a) Lasalocid sodium stock soln. — 100 /xg/mL. Accurately 
weigh suitable amt Lasalocid Na Ref. Std (available from 
Hoffmann-La Roche Inc.) and dil. to appropriate vol. with an- 
hyd. MeOH. 

(b) Std response line. — Dil. aliquots stock soln, (a), using 
anhyd. MeOH and H 2 0, to obtain concns of 0.25, 0.5, 1.0, 
2.0, and 4.0 [xg/mL in 25% MeOH (v/v). Ref. concn is I 
[xg/mL. Solns are stable <1 month at room temp. 

C. Stock Culture and Preparation of Inoculum 

Prep, slant culture of Bacillus subtilis, ATCC 6633, on >1 
tube of agar medium A, 957.23A(a). Incubate 16-24 hr at 
37°. Wash growth from stock culture with ca 3 mL sterile distd 
H 2 0, transfer liq. to surface of 300 mL agar medium G, 
957.23A(g), in Roux bottle, and incubate 7 days at 37°. Wash 
growth from agar surface with ca 25 mL sterile distd H 2 into 
centrf. bottle. Heat suspension 30 min in 65° H 2 bath. Centrf., 
decant, and resuspend cells in ca 25 mL sterile distd H 2 0. 
Repeat centrfg and suspending 3 times, discarding H 2 wash- 
ings. Heat residual spores 30 min in 65° H 2 bath. Resuspend 
spores in ca 35 mL sterile distd H 2 0. Suspension may be kept 
1 yr at ca 5°. Before use, dil. suspension with sterile distd H 2 
(usually 1 + 50) to read 20% Ton spectrophtr at 530 nm; store 
<1 week at 5°. 



122 



Drugs in Feeds 



AOAC Official Methods of Analysis (1990) 



D. Plates 

Seed layer. — Use single inoculated agar layer. Before as- 
say, det. by prepn of trial plates optimum concn (usually 5 mL 
for each 100 mL seed agar) of dild suspension of B. subtilis, 
975. 60C, to be added to agar medium M to obtain zones of 
inhibition of adequate size (17.5 ± 2.5 mm with 1 .0 |mg/mL) 
and sharpness. For actual assay, add appropriate amt of sus- 
pension to agar medium M previously melted, adjusted to pH 
6.0, and cooled to 60°. Mix thoroly and add 6.0 mL to each 
plate. Distribute evenly and let harden on perfectly level sur- 
face. Prep plates 2.5-3 hr before use. 

E. Assay Solution 

(a) Premixes, 15%. — Accurately weigh 1.0 g premix, 
transfer to 200 mL vol. flask, add 100 mL MeOH, shake vig- 
orously 3 min, and dil. to vol. with MeOH. Dil. 4.0 mL of 
this diln to 100 mL with MeOH. Further dil. 3.0 mL of last 
diln with 22 mL MeOH and H 2 to 100 mL (1 mL = ca 1 
u.g lasalocid Na/mL 25% MeOH). 

(b) Final feed, 0.0075%.— Weigh 20 g mash feed or pel- 
lets ground to pass No. 20 sieve and transfer to 500 mL vol. 
flask. Add 12 mL pH 4.7 buffer (975.61C(a)) and wet feed 
thoroly. Immerse flask 5 min in 70° H 2 bath. Cool to room 
temp. Add 200 mL EtOAc, stopper, and shake mech. 10 min. 
Centrf. ca 100 mL EtOAc ext 10 min at 2000 rpm. Pipet 60 
mL clear EtOAc ext into 200 mL vol. flask, add 8 mL 1.57V 
HC1, and shake 10 min. Let layers sep., transfer EtOAc layer 
to 100 mL g-s centrf. tube, and centrf. 10 min at 2000 rpm. 
Pipet 40 mL clear EtOAc ext into another 100 mL g-s centrf. 
tube and add 2 mL 40% NaOH soln. Stopper and shake briefly 
by hand, add 8 g anhyd. Na 2 S0 4 , and shake again. Centrf. 10 
min at 2000 rpm and decant 25 mL clear supernate into 50 
mL g-s graduate. Evap. all EtOAc under stream of N with 
graduate immersed in 60° H 2 bath. Dissolve residue in 5 mL 
hexane, add exactly 25 mL 75% MeOH (v/v), stopper, and 
shake vigorously 1 min. Transfer to 125 mL separator and let 
stand ca 1 hr. Withdraw lower (MeOH) layer into 25 mL beaker, 
pipet 5 mL into 50 mL vol. flask, and dil. to vol. with 19.4% 
(v/v) MeOH. 

F. Assay 

Using lasalocid Na std response line, assay soln, and plates, 
proceed as in 957.23E-F, incubating at 35 ± 1°. Calc. L and 
H and fit straight line by simplified least square method, 
957.23E. 

Refs.: JAOAC 57, 978(1974); 58, 941(1975); 59, 398, 
1286(1976). 

CAS-25999-31-9 (lasalocid) 



967.40 Lincomycin in Feeds 

Microbiological Method 
First Action 1967 
Method I 

(Applicable to feeds contg ^3.63 g/ton) 

A. Standard Solutions 

(a) Lincomycin stock soln. — Accurately weigh ca 40 mg USP 
Lincomycin. HO Ref. Std and dissolve in enough pH 8 buffer, 
957.23B(b), to give concn of exactly 100 jxg lincomycin base/ 
mL. Store <30 days at 2-10°. 

(b) Std response line. — Dil. aliquots stock soln, (a), with 
enough pH 8 phosphate buffer, 957.23B(b), to obtain concns 
of 0.2, 0.4, 0.8, 1.6, and 3.2 |mg lincomycin base/mL. Ref. 
concn is 0.8 |xg/mL. 



B. Plates 

(a) Base layer. — Add 10 mL melted agar medium E to ster- 
ile petri dishes, distribute evenly, and let harden on perfectly 
level surface. 

(b) Seed layer. — Before assay, det. by prepn of trial plates 
optimum concn of organism suspension of S. lutea (usually 
0.02-0.05% of suspension prepd as in 957.23D(e)(7) or 0.2- 
1% as in 957. 2310(e)(2)) to be added to agar medium J to 
obtain zones of inhibition of adequate size (16 mm ± 10% 
with 0.8 |xg/mL) and sharpness. For assay, add appropriate 
amt of organism suspension to agar medium J previously melted 
and cooled to 48°. Mix thoroly and add 4.0 mL to each plate 
contg base layer. 

C. Assay Solution 

Obtain and prep, sample as in 965.16 and 950.02. Accu- 
rately weigh ca 10 g ground sample and transfer to 250 mL 
g-s, r-b flask, add 20 mL H 2 0, and shake 10 min on wrist- 
action shaker. Add 50 mL 0.1 N HCl-MeOH (1 + 4) and shake 
10 min. Filter thru Whatman No. 4 paper, using 42 mm buch- 
ner and 500 mL flask. Repeat extn twice, using 50 mL HCl- 
MeOH each time. (Do not add more H 2 0.) Alternatively, con- 
duct extns in 250 mL centrf. bottle and centrf. to clarify. 

Transfer combined filtrates to 500 mL r-b flask and evap. 
to 15-20 mL, using rotary evaporator. (Do not heat >60°.) 
Transfer aq. ext to 125 mL separator. Rinse flask successively 
with 10 mL Skelly solve B, 951.01 A(o), 7-8 mL phosphate 
buffer, 957.236(b), and 10 mL Skellysolve B. Add all rins- 
ings to separator, shake, and let sep. Drain aq. phase, ext 
Skellysolve B twice with 7-8 mL buffer, and adjust combined 
exts to pH 8.0 with dil. NaOH soln. Adjust vol. with pH 8 
buffer to 0.6-1.0 u>g lincomycin base/mL. 

D. Assay 

Using lincomycin std response line and assay soln, proceed 
as in 957.23E-F, incubating at 32°. 

Refs.: JAOAC 50, 442(1967); 61, 1107(1978). 

CAS-154-21-2 (lincomycin) 



978.31 Lincomycin in Feeds 

Microbiological Method 
First Action 1978 
Method II 

(Applicable to feeds, feed supplements, and vitamin-mineral 
premixes contg 20-2600 g/ton) 

A. Standard Solutions 

Std response line (for monolayer plates). — Dil. aliquots stock 
soln, 967.40A(a), with enough pH 8 phosphate buffer, 
957.23B(b), to obtain concns of 0.15, 0.3, 0.6, 1.2, and 2.4 
|xg lincomycin base/mL. Ref. concn is 0.6 |mg/mL. 

B. Plates 

Monolayer. — Proceed as in 967.40B(b), except zones of in- 
hibition should be 18.5 mm ± 10% with 0.6 |xg/mL; add 7,5 
mL final mixt. to each plate, distribute evenly, and let harden 
on perfectly level surface. 

C. Assay Solution 

Obtain and prep, sample as in 965.16 and 950.02. 

(a) For 20-80 g/ton.— Accurately weigh ca 10-20 g ground 
sample (see Table 978.31), and transfer quant, to 250 mL centrf. 
bottle. Add 75.0 mL 0.1N HCl-M'eOH (1 + 4), and shake 20 
min on mech. shaker. Centrf. 5 min at ca 2500 rpm to clarify. 



AOAC Official Methods of Analysis (1990) 



Monensin 123 



Decant supernate into 250 mL separator, add 75 mL hexane, 
and shake moderately 1 min. Let layers sep. >15 min. Drain 
lower aq. layer into 100 mL beaker, and pipet 2.5-5 mL ali- 
quot into 50 mL mixing cylinder. Adjust vol. to ca 40 mL 
with pH 8 phosphate buffer, 957.236(b), and add 1 drop 4/V 
NaOH. Stopper and shake vigorously. If necessary, adjust to 
pH 8 and dil. to 50 mL with p.H 8 buffer. Final concn should 
be 0.5-0.8 jutg lincomycin base/mL. 

(b) For 80-2600 g/ton. — Accurately weigh 4-6 g ground 
sample (see Table 978.31), and transfer quant, to 250 mL centrf. 
bottle. Add 50.0 mL 0AN HCl-MeOH (1 + 4), shake 20 min 
on mech. shaker, and add 50 mL pH 8 buffer. Shake mech. 
5 min and centrf. 5 min at ca 2500 rpm to clarify. Dil. 1.0 
mL aliquot with pH 8 buffer to 0.4-0.8 jULg lincomycin base/ 
mL. 

D. Assay 

Using lincomycin std response line and assay soln, proceed 
as in 957.23E-F, incubating at 32°, except use 2 plates instead 
of 3 and 6 readings instead of 9. Av. of all 24 readings of ref. 
concn from 8 plates is correction point for response line. 

Refs.: JAOAC 50, 442(1967); 61, 1 107(1978). 

CAS-154-21-2 (lincomycin) 



972.56 Monensin in Feeds 

Microbiological Method 
First Action 1972 

(Applicable to feeds contg >90 g/ton) 

A. Standard Solutions 

(a) Monensin stdsolns. — (1) Stock soln. — 1 mg/mL. Ac- 
curately weigh enough Monensin Na Salt Ref. Std (Elanco 
Products Co.) into 100 mL vol. flask to give concn of 1 mg 
free acid/mL. Dil. to vol. with anhyd. MeOH. Store at 5°; 
discard after 2 weeks. (2) Working soln. — 100 juug/ mL. Dil. 
.1 mL stock soln to 10 mL with aq. MeOH (1 + 1). Prep. 
fresh daily. 

(b) Std response line. — Dil. aliquots of working soln, (a)(2), 
with enough aq. MeOH (I + 1) to obtain concns of 0.25, 0.5, 
1.0, and 2.0 jixg monensin/mL. Ref. concn is 0.5 u,g/mL. 

B. Plates 

Seed layer. — Use single inoculated agar layer. Before as- 
say, det. by prepn of trial plates optimum concn (usually 0.5 
mL for each 100 mL seed agar) of suspension of B. subtilis, 
957.23D(d), to be added to agar medium L to obtain zones of 
inhibition of adequate size (17.5 ± 2.5 mm with 0.5 (xg/mL) 
and sharpness. Before use, dil. suspension with sterile H 2 to 
read 20% T on spectrophtr at 530 nm; prep, dild suspension 
daily. For actual assay, add appropriate amt of suspension to 
agar medium L previously melted and cooled to 48-50°. Mix 



thoroly and add 6.0 mL to each plate. Cover and refrigerate 
>1 hr before use. 

C. Assay Solution 

Place glass wool plug at bottom of 19 mm id X 500 mm 
chromatgc tube and add ca 75 mm alumina, 961.23C(a), with 
gentle tapping. Accurately weigh sample (20 g finished feed, 
5 g premix) and add to column. Elute column with MeOH- 
H 2 (9 + 1), using 200 mL vol. flask as receiver. Do not 
restrict flow. Collect 200 mL eluate, mix, and dil. with aq. 
MeOH (1 + 1) to 0.5 |xg monensin/mL. Designate soln ob- 
tained as assay soln. 

D. Assay 

Use 10 seeded agar plates for std curve. Place 4 stainless 
steel cylinders, 957.23C(a), on each plate at 90° intervals. Fill 
1 cylinder on each plate with different concn of std soln, (b), 
and incubate all plates 16-18 hr at 35-37°. Measure diams 
of zones of inhibition. Calc. av. zone diam. at each std concn 
and fit straight line by simplified least squares method, 957. 23E. 

E. Determination 

Use 5 plates for each assay soln and align cylinders at 90° 
intervals. On each plate fill alternate cylinders with ref. concn 
and fill other cylinders with assay soln. Incubate plates 16- 
18 hr at 35-37° and measure diams of zones of inhibition to 
nearest 0.1 mm. Average 10 readings of ref. concn and 10 
readings of assay soln. Proceed as in 957. 23F. 

Ref.: JAOAC 55, 718(1972). 

CAS- 17090-79-8 (monensin) 



976.37 



Monensin in Feeds 

Turbldimetric Method 

First Action 1976 



A. Apparatus and Reagents 

(a) Assay broth. — In 500 mL erlenmeyer, dissolve 9.0 g 
low K ion medium (N-Z Case, Humko-Sheffield Chemical, 
PO Box 398, Memphis TN 38101), 3.0 g yeast ext, and 8.0 
g glucose in 100 mL H 2 while heating to bp. Cool imme- 
diately, and add to bottle contg 1500 mL sterile H 2 0, 4.5 mL 
10% soln of polysorbate 80, and 1 2.5 mL citrate buffer soln, 
(c). pH should be 5.2 ± 0.1; adjust if necessary. 

(b) Automated turbldimetric system. — Autoturb® (Elanco 
Products Co.), or equiv. System pipets samples into assay tubes, 
dils with inoculated broth, incubates, and measures turbidity. 

(c) Citrate buffer soln. — pH 4.0. Dissolve 105 g citric 
acid.fLO, 142 g Na citrate. 2H 2 0, and 1.9 g KC1 in H 2 and 
dil. to 1 L with H 2 0. 

(d) Monensin std solns. — (J) Stock soln. — 1 mg free acid/ 
mL. Accurately weigh enough Monensin Na Salt Ref. Std 
(Elanco Products Co., Department MC757) into 100 mL vol. 



Table 978.31 Examples of Sample Size and Dilution of Extract 



Assay 


Feed Level of 


Lincomycin, 


Sample 


Total 






Final Concn, 


Soln. 






Extn Vol., 


Aliquot 


Final Vol., 


978.31 C 


g/ton 


^g/g 


Size, g 


mL 


Vol., mL 


mL 


j^g/mL 


(a) 


20 


22 


20.0 


75 


5.0 


50 


0.59 


(a) 


40 


44 


10.0 


75 


5.0 


50 


0.59 


(a) 


80 


88 


10.0 


75 


2.5 


50 


0.59 


(b) 


80 


88 


6.0 


100 


1.0 


10 


0.53 


(b) 


140 


154 


6.0 


100 


1.0 


15 


0.62 


(b) 


200 


220 


6.0 


100 


1.0 


25 


0.53 


(b) 


400 


440 


6.0 


100 


1.0 


50 


0.53 


(b) 


1000 


1100 


4.0 


100 


1.0 


100 


0.44 


(b) 


2600 


2860 


4.0 


100 


1.0 


200 


0.57 



124 



Drugs in Feeds 



AOAC Official Methods of Analysis (1990) 



flask to give concn of 1 mg free acid/mL. Add MeOH to dis- 
solve salt, dil. to vol. with MeOH, and stopper tightly. (Soln 
is stable at room or refrigerator temp. 1 month, except for gain 
in potency from loss of MeOH. Warm refrigerated soln to room 
temp, before pipetting.) (2) Intermediate soln. — 10 ixg/mL. 
Dil. 1.0 mL stock soln to 100 mL with MeOH. (3) Autoturb 
working solns. — Using pipets and vol. flasks, prep, solns contg 
0.25, 0.50, 0.75, and 1 .0 'p,g/mL MeOH. (4) Manual working 
solns. — Dil. stock soln to 10.0 \Lg/mL with MeOH. Prep, std 
solns of 0.0, 0.05, 0.10, 0.15, and 0.20 fxg/mL by adding 
appropriate vols of 10.0 \xg/mL soln to vol. flasks, adding 
enough MeOH so that final MeOH concn is 20% in each flask, 
and adjusting to vol. with H 2 0. 

B. Preparation of Inoculum 

Use Streptococcus faecalis (ATCC 8043) as assay organism 
in either frozen suspension or freshly grown inoculum. Prep, 
latter by inoculating flask of broth medium A, 957.23 A(n) 
(Difco No. 3 Broth), in afternoon and leaving flask overnight 
at room temp. Use 10-20 mL inoculum/L assay medium. Let 
assay medium inoculated with frozen suspension stand at room 
temp. 45 min before using. (This treatment prevents drift within 
test.) 

C. Preparation of Samples 

Grind >100 g feed sample, and mix. Accurately weigh 10.0 
g representative well ground sample, and transfer to 4 oz (125 
mL) jar fitted with Al foil-lined cap. Add 100.0 mL MeOH 
and tightly cap jar. Shake well and let stand overnight or >8 
hr. Approx. 30 min before dilg, shake once more and let solids 
settle. 

(a) Autoturb system measurement. — Dil. 1 .0 mL ext to 20.0 
mL with MeOH to prep, soln to be dild by system. 

(b) Manual measurement. — Dil. ext 100-fold (1 + 99) and 
66-fold (3 + 197) to obtain approx. assay concns of 0.10 and 
0.15 |ULg/mL by adding aliquots of sample to sep. vol. flasks, 
adding MeOH to 20% final concn, and dilg to vol. with H 2 0. 
These solns are stable >1 week at room temp. 

D. Assay 

(a) Autoturb system measurement. — Place assay tube car- 
rier in diluter unit. Place total of 20 sample tubes in sample 
turntable (including 5 tubes contg the 4 std levels and 1 tube 
with MeOH) in middle of test series. Fill remaining places in 
turntable with samples dild to estd concn of 0.6 jig/mL, When 
diluter unit has processed tubes, place carrier in 37.5° H 2 
bath 3-4 hr or until turbidity of tube measures 40% T or 
slightly less. Stop growth by heating carrier with tubes in 80° 
H 2 bath 1-2 min. Cool and read at 650 nm. 

(b) Manual measurement. — (1) Std curve. — Prep, series 
of culture tubes, in triplicate, by adding 0.5 mL of each work- 
ing std soln to sep. tubes. Add 9.5 mL inoculated assay broth 
to each tube. (2) Samples. — Prep. 2 dilns for each sample to 
contain ca 0.1 and 0.15 |xg monensin activity /mL soln. Add 
0.5 mL of each diln to 3 sep. tubes, and add 9.5 mL inoculated 
assay broth to each tube. Sample levels are c a 0.05 and 0.075 
jjig/tube. 

Incubate std and sample tubes 4-5 hr at 37.5° or until tur- 
bidity of control std tubes (0.0 monensin) measures ca 30% T 
at 650 nm. Terminate growth by heating tubes >2 min at 80°. 
Cool tubes, and measure turbidity of each tube at 650 nm. 

E. Preparation of Standard Curve 

(a) Autoturb system measurement. — Prep. 2 dose-response 
curves, 1 for 0.1 mL loop and other for 0.15 mL loop. Prep. 
graph of log % T against concn. Use av. turbidity of each pair 
of tubes in prepg std curves. Assay each sample in duplicate 
at 2 concns, 0.10 mL and 0.15 mL sample. Average readings 



from 0.10 mL loop for sample and obtain monensin equiv. by 
interpolation from 0.10 mL std curve. Repeat for 0.15 mL 
loop and use 0.15 mL std curve. 

(b) Manual measurement. — Prep, only single std curve. 
Prep, graph of log % T against concn. Average 3 readings 
obtained for each diln of sample. Obtain monensin equiv. for 
each sample diln by interpolation from std curve. 

F. Calculations 

(a) Autoturb system measurement. — Average 2 readings of 
0.10 mL vols for feed sample and obtain monensin equiv. by 
interpolation from 0.10 mL std curve (/*,). Repeat for 0.15 
mL sample vols to obtain P 2 . 

|mg Monensin activity /mL = (Pi + Pi)/2 - P A 

fig Monensin/g feed = P A X 200 

g Monensin activity (free acid/ton feed) 

= P A x 200 x 0.908 

(b) Manual measurement. — Multiply av. monensin equiv. 
for each of the 2 sample dilns (M, = 0. 10 mL diln, M 2 — 0. 15 
mL diln) by its respective diln factor (e.g., 100 and 66). Av- 
erage the 2 values to obtain potency of original material (P A ). 

(xg Monensin activity /mL = (M ] + M 2 )/2 = P A 

u,g Monensin/g feed = P A x 10 

g Monensin activity (free acid/ton feed) 

= P A x 10 x 0.908 

Refs.: JAOAC 55, 114(1972); 60, 179(1977). 

CAS- 17090-79-8 (monensin) 



970.89 



Neomycin in Feeds 

Microbiological Method 

First Action 1970 



(Applicable to feeds contg >28 g neomycin base /ton; soybean 
content >40% reduces accuracy of method.) 

A. Standard Solutions 

(a) Stock soln.— Dry USP Neomycin Sulfate Ref. Std 3 hr 
in vac. oven at <5 mm (0.66 kPa). Accurately weigh enough 
dried std (10-50 mg) and dissolve in Tris buffer, 957.23B(t), 
to give concn of 100 jxg neomycin base/mL. (Neomycin sul- 
fate equiv. to neomycin base is given on container.) Store <4 
weeks at 2-10°. 

(b) Std response line. — Dil. aliquots of stock soln (a) with 
enough inactivated feed ext, 970.89C(b), to obtain 0.50, 0.75, 
1,13, 1.69, and 2.53 \xg neomycin base/mL. Prep, std re- 
sponse line for each feed sample. 

a Plates 

(a) Base layer. — Add 10 mL melted agar medium K to petri 
dishes, distribute evenly, and let harden on perfectly level sur- 
face . 

(b) Seed layer. — Add appropriate amt (usually 0.5- 2%) 
broth culture of S. epidermidis, 957.23D(f), to agar medium 
K previously melted and cooled to 48°. Mix thoroly and add 
4.0 mL to each plate contg base layer. Distribute agar evenly 
by tilting plates from side to side with circular motion and let 
harden. Use plates same day prepd. Zone of inhibition of 18 
mm ± 15% should be obtained with 1.13 jxg/mL. 

C. Assay Solution 

(a) Preparation of sample. — Obtain and prep, sample as in 
965.16 and 950.02. Weigh 20 g sample into 500 mL r-b flask. 



AOAC Official Methods of Analysis (1990) 



Novobiocin 



125 



Table 970.89 


Dilution of Extract 






Neomycin 
Base, 

g/ton 


Sample 
Extract, 
Diln I 


Neomycin 

Base, Final 

Concn, 

^g/mi_ 


Inactivated 
Extract, 
Diln II 


140 
70 
35 


1 to 20 
1 to 10 
1 to 10 


1.54 
1.54 
0.77 


10 to 200 
20 to 200 
20 to 200 



Add 100.0 ml NaCl-CaCl 2 soln, 957.23B(n), and shake 15 
min on wrist-action shaker. Transfer to 250 mL centrf . bottle 
(do not rinse), centrf. 15 rnin at 1800-2000 rpm, and decant 
supemate into beaker. Transfer 20 mL aliquot to 100 mL beaker 
and set aside to prep, std response line diluent. Using pH me- 
ter, adjust remaining portion with HO to pH 2.0. Wait >5 
min; then readjust to pH 8.0 with ION NaOH. (High concns 
of acid and base are used to avoid significant changes in vol.) 
Centrf. 30-35 mL 15 min at 1800-2000 rpm. Dil. ext soln 
with Tris buffer according to neomycin content in feed as in 
Table 970.89 (diln I). 

(b) Preparation of std response line diluent. — Inactivate the 
20 mL aliquot from (a) by adjusting to pH 4.5-4.7 with 2N 
HC1. Add 1.5 mL fresh 5.25% NaOCl soln, 957.23B(o), and 
heat 45 min in boiling H 2 bath, stirring thoroly at least every 
10 min during heating period. Cool to room temp., adjust to 
pH 8.0 with 3.5N NaOH, and dil. to 20 mL with H 2 0. Dil. 
inactivated ext with Tris buffer, 957.236(1), according to neo- 
mycin content in feed as in diln II column of Table 970.89. 

Use dild soln to prep, std response line solns. 

D. Assay 

Using neomycin std response line, assay soln, and plates, 
proceed as in 957.23E-F, incubating at 32-35°. Result may 
be calcd as neomycin sulfate by multiplying neomycin base by 
1.428 (1.0 mg neomycin sulfate is equiv. to 0.7 mg neomycin 
base). 

Ref.: JAOAC53, 60(1970). 

CAS- 1404-04-2 (neomycin) 



962.25 Novobiocin in Feeds 

Microbiological Method 

First Action 1962 
Final Action 1965 

(Applicable to feeds contg ^350 ppm) 

A. Standard Solutions 

(a) Novobiocin stock soln. — Dry USP Novobiocin Ref. Std 
3 hr at 60° in vac. oven at <5 mm (0.66 kPa). Accurately 
weigh ca 30 mg dried std, dissolve in 10 mL absolute alcohol, 
and dil. with enough pH 8 phosphate buffer, 957.236(b), to 
give concn of 1 mg/mL. Store <3 weeks at 2-10°, 

(b) Std response line for feed supplements. — Dil. aliquots 
of stock soln, (a), with enough pH 6 buffer, 957.23B(f), to 
obtain concns of 1.9, 2.4, 3.0, 3.8, and 4.7 u^g/mL. Ref. 
concn is 3.0 |ULg/mL. 

(c) Std response line for finished feeds. — Dil. aliquots of 
stock soln, (a), with enough pH 6 buffer, 957.236(f), to ob- 
tain concns of 0.128, 0.16, 0.20, 0.25, and 0.312 [xg/mL. 
Ref. concn is 0.20 |xg/mL. 

0. Plates 

(a) For feed supplements. — (7) Base layer. — Add 21 mL 
melted agar medium C to sterile petri dishes, distribute evenly, 
and let harden on perfectly level surface. 



(2) Seed layer. — Before assay, det. by prepn of trial plates 
optimum concn of organism suspension of S. lutea (usually 
0.2-0.5% of suspension prepd as in 957. 230(e)(7) or 2-5% 
as in 957.23D(e)(2)) to be added to agar medium C to obtain 
zones of inhibition of adequate size (14 mm ± 10% with 3.0 
|xg/mL) and sharpness. For actual assay, add appropriate amt 
of organism suspension to agar medium C, previously melted 
and cooled to 48°. Mix thoroly and add 5.0 mL to each plate 
contg base layer. 

(b) For final feed. — (/) Base layer. — Prep, as in (a) (7), 
using 15 mL melted agar medium C. 

(2) Seed layer. — Add appropriate amt (usually 0.5- 2%) 
broth culture of S. epidermidis, 957.23D(f), to agar medium 
C previously melted and cooled to 48°. Mix thoroly and add 
5 mL to each plate contg base layer. Zone of 14 mm ± 10% 
should be obtained with 0.20 (xg/mL. 

C. Assay Solution 

(a) For feed supplements containing 50 mg/g. — Obtain and 
prep, sample as in 965.16 and 950.02. Accurately weigh suit- 
able size sample and add enough absolute alcohol to give estd 
concn of 2 mg/mL. Let stand 30 min, shaking occasionally. 
Add equal vol. pH 8 phosphate buffer, 957.236(b), and mix. 
Dil. to estd concn of 3 u.g/mL with pH 6 buffer, 957.236(f), 
and use as assay soln. 

(b) For final feed containing not less than 350 pg/g. — Ob- 
tain and prep, sample as in 965.16 and 950.02. Weigh 1 g 
sample into 50 mL g-s graduate; ext twice with 20 mL EtOAc, 
shaking vigorously 2 min. Decant supernate into second 50 
mL g-s graduate. Dil. to 50 mL with EtOAc. Transfer 2.0- 
4.0 mL aliquot to 100 mL vol. flask, add 5.0 mL pH 8 phos- 
phate buffer, 957.236(b), and mix thoroly. Dil. to vol. with 
pH 6 buffer, 957.236(f), and shake vigorously to dissolve all 
EtOAc. Final concn of novobiocin should be 0.15-0.30 jxg/ 
mL. 

D. Assay 

Using proper novobiocin std response line, assay soln, and 
plates, proceed as in 957.23E-F, incubating at 32-35°. 

Ref.: JAOAC 45, 310(1962). 

CAS-303-81-1 (novobiocin) 



974.48 Nystatin in Feeds 

Microbiological Method 
First Action 1974 

(For feeds contg >50 g/ton) 

A. Standard Solutions 

(a) Nystatin stock soln. — Dry ca 25 mg USP Nystatin Ref. 
Std 2 hr at 40° in vac. oven at <5 mm (0.66 kPa). Accurately 
det. dry wt and add enough MeOH to give concn of exactly 
500 units/mL. Dissolve by shaking on mech. shaker 0.5 hr 
(soln may be slightly hazy). Prep, fresh daily. (1 g nystatin = 
2,800,000 units.) 

(b) Std response line. — Dil. aliquots stock soln, (a), with 
enough inactivated feed ext, 974.48C(b), to obtain concns of 
10, 20, and 40 units/mL. Prep, std response line for each feed 
sample. Ref. concn is 20 units/mL. 

B. Plates 

Seed agar. — Use single inoculated agar layer. Before assay, 
det. by prepn of trial plates optimum concn of organism sus- 
pension of Sacch. cerevisiae (usually 2.5% of suspension prepd 
as in 957.23D(g)(7)), to be added to agar medium I, 957.23A(i), 
to obtain zones of inhibition of adequate size (18 mm ± 10% 



126 



Drugs in Feeds 



AOAC Official Methods of Analysis (1990) 



for 20 units/mL) and sharpness. For actual assay, add appro- 
priate amt of organism suspension to agar medium I, previ- 
ously melted and cooled to 48°. Mix thoroly and add 10 mL 
to each sterile petri dish. 

C. Assay Solution 

(a) Preparation of sample. — Obtain and prep, sample as in 
965.16 and 950.02. Weigh amt sample contg ca 50 g nystatin/ 
ton (ca 7700 units total), into 500 mL erlenmeyer. Add 150 
mL anhyd. MeOH, mix thoroly by hand, and then shake 1 hr 
on rotary shaker. Centrf. briefly. Dil. 4 parts ext with 6 parts 
10% phosphate buffer, 957.23B(e). Set aside 10 mL as sample 
test soln and use remainder to prep, response line diluent. 

(b) Preparation of std response line diluent. — Measure vol. 
of remaining diid ext soln from (a). Place soln in cotton-plugged 
erlenmeyer having twice capacity of vol. to be contained, and 
autoclave 15 min at 121° (slow exhaust). Cool to room temp, 
and restore to original vol. by adding anhyd. MeOH. Mix tho- 
roly and use dild soln to prep, std response line solns. 

D. Assay 

Use 10 plates for each sample. On each plate, fill alternate 
cylinders, each with I of std response line solns, and fill other 
3 cylinders with sample test soln. Incubate plates overnight at 
30-37°; then measure diams of zones of inhibition to nearest 
0.1 mm. Calc. av. zone diam. at each std concn; plot values 
on semilog graph paper and draw line as in 957. 23E. Calc. 
av. zone diam. of sample test soln. Using this value, det. po- 
tency of antibiotic from std response line. 

Ref.: JAOAC 57, 536(1974). 

CAS- 1400-6 1-9 (nystatin) 



974.49 Oleandomycin in Feeds 

Microbiological Method 

First Action 1974 
Final Action 1975 

(For feeds contg >2 g/ton) 

A. Standard Solutions 

(a) Oleandomycin stock soln. — 250 |xg/mL. Accurately 
weigh Oleandomycin Chloroform Adduct Ref. Std (available 
from Pfizer, Inc., 235 E. 42nd St, New York, NY 10017) or 
USP Ref. Std. Dissolve in ca 5 mL MeOH and dil. with enough 
pH 8.0 phosphate-bicarbonate buffer, 957.236(a), to give concn 
of 250 |xg/mL. Prep, fresh daily. 

(b) Intermediate std soln. — 5 |xg/mL. Dil. 5.0 mL stock 
soln to 250 mL in vol. flask with pH 8.0 phosphate-bicarbon- 
ate buffer, 957.23B(a). 

(c) Std response line. — Dil. intermediate std soln with pH 
8.0 buffer, 957.23B(a), to obtain concns of 0.025, 0.05, 0.10, 
0.20, and 0.40 |xg/mL. Ref. concn is 0.10 fxg/mL. 

B. Assay Solution 

Obtain and prep, sample as in 965.16 and 950.02. Accu- 
rately weigh 20 g sample contg >2 g oleandomycin/ ton into 
500 mL boiling flask, add 200 mL pH 8.0 phosphate-bicar- 
bonate buffer, 957.238(a), and shake mech. 45 min. Let set- 
tle, decant into 50 mL centrf. tube, and centrf. 5 min at 2000 
rpm. Dil. supernate with addnl pH 8.0 buffer to approx. ref. 
concn of 0.1 u^g/mL. Use sample ext same day prepd. 

C. Plates 

Use single inoculated agar layer. Before assay, det. by prepn 
of trial plates optimum concn of organism suspension of 5. 
lutea (usually 0.05-0.1% of suspension prepd as in 



957.23D(e)(7)) to be added to agar medium J to obtain zones 
of inhibition of adequate size (16 mm ± 10% with 0.1 (xg/ 
mL) and sharpness. For actual assay, add appropriate amt of 
organism suspension to agar medium J previously melted and 
cooled to 48°. Mix thoroly and add 10.0 mL to each petri dish. 

D. Assay 

Using oleandomycin std response line, assay soln, and plates, 
proceed as in 957.23E-F, incubating plates at 37°, 

Refs.: JAOAC 56, 1149(1973); 57, 823(1974). 

CAS-3922-90-5 (oleandomycin) 



968.50 Oxytetracycline In Feeds 

First Action 1968 
Final Action 1970 

(Applicable to feeds contg >10 g/ton) 

A. Standard Solutions 

(a) Oxytetracycline stock soln. — Accurately weigh ca 40 mg 
Oxytetracycline USP Ref. Std and dissolve in enough 0.17V 
HC1 to give exact concn of 100 jxg oxytetracycline /mL. (1 |xg 
base is equiv. to 1.08 |xg of the hydrochloride.) Store in dark 
<5 days at 2-10°. 

(b) Std response line. — Dil. appropriate aliquots stock soln, 
(a), with enough pH 4.5 buffer, 957.23B(g), to obtain concns 
of 0.05, 0.10, 0.20, 0.40, and 0.80 \xg oxytetracycline/mL. 
Ref. concn is 0.20 jxg/mL. 



Microbiological Method I 

(Applicable to >220 mg/lb) 

B. Assay Solution 

(Solv. losses may occur from evapn of volatile soJvs in open 

containers. Carefully measure and record vols, of solvs, and 

make appropriate mathematical corrections for any losses 

in vol.) 

Obtain and prep, sample as in 965.16 and 950.02. Using 
mortar and pestle or high-speed blender, grind 2 g sample with 
50 mL acid-MeOH, 957.23B(i), and transfer mixt. to 100 mL 
centrf. tube. Wash mortar and pestle or blender jar with 50 
mL acid-MeOH and combine washings with ext in centrf. tube. 
Shake well 5 min. Centrf. ca 15 min at 2000 rpm. Remove 
10 mL clear soln and adjust to pH 4.5 with 1/V NaOH. Dil. 
adjusted soln with enough pH 4.5 buffer, 957.236(g), to ob- 
tain estd concn of 0.20 fjig/mL. Designate as assay soln. 

C. Assay 

Using oxytetracycline std response line and assay soln, and 
chlortetracycline plates, 967.39B(b), proceed as in 957.23E- 
F, incubating at 30°. 



Microbiological Method II 

(Applicable to <220 rag/lb) 

D. Plates 

Use single inoculated agar layer. Before assay, det. by prepn 
of trial plates optimum concn (usually 0.03-0.10%) of stock 
suspension of B. cereus, 957.23D(c), to be added to agar me- 
dium D, 957.23A(d), to obtain zones of inhibition with as lit- 
tle as 0.05 |xg oxytetracycline/mL. Zone of 18 mm ± 10% 



AOAC Official Methods of Analysis (1990) 



Procaine Penicillin 



127 



should be obtained with 0.20 (xg/mL. For actual assay add 
appropriate amt of inoculum to agar medium D previously 
melted and cooled to 48°. Mix thoroly, and add 9.0 mL to 
each plate. 

E. Assay Solution 

(Solv. losses may occur from evapn of volatile solvs in open 

containers. Carefully measure and record vols of solvs, and 

make appropriate mathematical corrections for any losses 

in vol.) 

Accurately weigh 20 g ground finished feed into 250 mL 
extn flask, add 100 mL acid-MeOH, 957.23B(i), stopper, and 
shake mech. 5 min. Centrf. ca 5 min at 2000 rpm. Remove 
20 mL supernate and adjust to pH 4.5 with 1W NaOH. Dil. 
adjusted soln with enough pH 4.5 buffer, 957.23B(g), to ob- 
tain estd concn of 0.20 jxg/mL and filter thru Whatman No. 
2V paper, or equiv. Designate as assay soln. 

F. Assay 

Using oxytetracycline std response line, assay soln, 968.50E, 
and plates, proceed as in 957.23E-F, incubating at 28-30°. 
Calc. result as oxytetracycline. HC1 by multiplying oxytetra- 
cycline base by 1.08. 

Ref.: JAOAC 51, 548(1968). 

CAS-79-57-2 (oxytetracycline) 



967.41 Procaine Penicillin in Feeds 

Microbiological Method 

First Action 1967 
Final Action 1968 

(Applicable to feeds contg ^1.5 g/ton) 

A. Standard Solutions 

(a) Penicillin stock soln. — Accurately weigh, in atm. of 
<50% relative humidity, ca 30 mg USP Potassium or Sodium 
Penicillin G Ref. Std. Dissolve in enough pH 6 buffer, 
957.23B(f ), to give known concn of 100-1000 units/mL. Store 
in dark <2 days at 2-10°. 

(b) Std response line. — Dil. appropriate aliquots of stock 
soln, (a), with enough pH 6 buffer, 957.23B(f), to obtain concns 
of 0.0125, 0.025, 0.05, 0.10, and 0.20 unit/mL. Ref. concn 
is 0.05 unit/mL. 

B. Plates 

(a) Base layer. — Add 10 mL melted agar medium A to sterile 
petri dishes, distribute evenly, and let harden on perfectly level 
surface. 

(b) Seed layer. — Before assay, det. by prepn of trial plates 
optimum concn of organism suspension of S. lutea (usually 
0.2-0.5% of suspension prepd as in 957.23D(e)(7) or 2-5% 
as in 957.230(e)(2)) to be added to agar medium B to obtain 
zones of inhibition of adequate size (19 mm ± 10% with 0.05 
unit/mL) and sharpness. 

For actual assay, add appropriate amt of organism suspen- 
sion to agar medium B previously melted and cooled to 48°. 
Mix thoroly and add 4.0 mL to each plate contg base layer. 

C. Assay Solution 

(Solv. losses may occur from evapn of volatile solvs in open 
containers. Carefully measure and record vols of solvs, and 
make appropriate mathematical corrections for any losses in 

vol.) 



Obtain and prep, sample as in 965.16 and 950.02. Vary amts 
of sample and extractant according to penicillin content of feed 
as follows: 



Penicillin Content 



Sample 
Size, g 



Vol. 

Extractant, 

mL 



>100 g/lb 


1 


100 


1-100 g/lb 


3 


100 


0.1-1 g/lb 


10 


100 


<0.1 g/lb (200 g/ton) 


50 


200 



Ext appropriate amt of sample with pH 6 buffer-acetone ex- 
tractant, 957.23B(k), in suitable container, using either wrist- 
action or reciprocating mech. shaker 30 min or high-speed 
blender 2 min; let settle and decant supernate. Centrf. if more 
than slightly turbid. Dil. aliquot of supernate with enough pH 
6 buffer, 957.238(f), to obtain estd concn of 0.05 unit/mL. 

D. Assay 

Using penicillin std response line, assay soln, and plates, 
proceed as in 957.23E-F, incubating at 26-32°. Calc. result 
in terms of units or wt (1 mg Na penicillin G = 1667 units; 
1 mg K salt - 1595 units; 1 mg procaine salt = 1009 units). 

E. Identity 

To aliquot of supernate assay soln, 967.41 C, add enough 
penicillinase soln to inactivate penicillin by incubating mixt. 
1 hr at 37°. Further dil. with enough pH 6 buffer, 957.238(f), 
to give same diln factor as in 967. 41C. Assay as in 967. 41D. 
Absence of zone of inhibition indicates that activity in sample 
is due to penicillin. (See 962.14E.) 

Ref.: JAOAC 50, 450(1967). 

CAS-54-35-3 (penicillin G procaine) 



973.81 Spectinomycin in Feeds 

Microbiological Method 
First Action 1973 

(Applicable in presence of lincomycin to feeds contg 5:18 
g/ton) 

A. Standard Solutions 

(a) Spectinomycin stock soln. — 1 mg spectinomycin base/ 
mL. Accurately weigh amt of Spectinomycin. HC1.5H 2 Ref. 
Std (available from Agricultural Division, The Upjohn Co.) 
and dissolve in enough H 2 to give concn of exactly 1.0 mg 
spectinomycin base/mL. Store in dark <30 days at 2-10°. 

(b) Std response line. — Dil. stock soln with Tris buffer, 
957.238(1), to obtain concn of 100 fxg/mL. Dil. this soln with 
Tris buffer to obtain 2.8, 4.4, 7.0, 11.0, and 17.4 \ig spec- 
tinomycin base/mL. Prep, daily. Ref. concn is 7.0 jxg/mL. 

B. Plates 

Before assay, det. by prepn of trial plates optimum concn 
of culture of E. coli, 957.23D(h) (usually ca 0.04%) to be 
added to agar medium F to obtain zones of inhibition of ad- 
equate size and sharpness. Zones of 13 ± 1 and 23 ± 1 mm 
should be obtained for 2.8 and 17.4 juug/mL, resp. 

For actual assay, add appropriate amt of culture to agar me- 
dium F, previously melted and cooled to 48°. Mix thoroly and 
add 7.0 mL to each plate. 

C. Assay Solution 

Weigh 20 g feed into 250 mL centrf. tube and add 100 mL 
acid-MeOH (20 mL 1/V HC1 dild to 1 L with MeOH). Shake 



128 



Drugs in Feeds 



AOAC Official Methods of Analysis (1990) 



15 min on wrist-action shaker, or equiv., centrf. 5 min at 2000 
rpm, and decant into 500 mL r-b flask. Repeat extn twice, 
combining exts in r-b flask. Evap. ext under vac. until all MeOH 
and most of H 2 have been removed. Do not exceed 60° and 
do not evap. to complete dryness. Add 30 mL Pb(OAc) 2 soln, 
957.23B(q), to flask, shake vigorously 2 min, and transfer quant, 
to 100 mL beaker. Rinse flask with 10-15 mL Tris buffer and 
add rinse to beaker. Adjust to pH 8.0 with 3.5N NaOH. Trans- 
fer quant, to 100 mL graduate, rinsing beaker with buffer, and 
dil. to 100 mL with buffer. Mix thoroly, and let sample stand 
30 min. Centrf. 30-50 mL 10 min at 2000 rpm. Decant su- 
pernate into test vials for storage (<2 days) until assay. Des- 
ignate soln obtained as assay soln. 

D. Assay 

Using spectinomycin std response line, assay soln, and plates, 
proceed as in 957.23E-F, incubating at 26°. 

g Spectinomycin /ton 

= (fig spectinomycin base/mL assay soln) X 4.54 
If zone size is plotted against g/ton instead of jmg base/mL 
working stds as follows, calcns are not necessary. 



\xg Base/mL 



g/ton 



2.8 


12.7 


4.4 


20.0 


7.0 


31.8 


11.0 


50.0 


17.4 


79.0 



Ref.: JAOAC 56, 834(1973). 
CAS-1695-77-8 (spectinomycin) 

971.49 Streptomycin in Feeds 

Microbiological Method 

First Action 1971 
Final Action 1973 

Method I 

(Applicable to feeds contg >30 g/ton) 

A. Standard Solutions 

(a) Streptomycin stock soln. — Dry ca 40 mg USP Strep- 
tomycin Sulfate Ref. Std 3 hr at 60° in vac. oven at <5 mm 
(0.66 kPa). Det. accurate dry wt and dissolve in enough H 2 
to give concn of exactly 100 u,g streptomycin base/mL. Store 
<30 days at 2-10°. 

(b) Std response line. — Dil. aliquots of stock soln, (a), with 
enough pK 8 buffer, 957.23B(b), to obtain concns of 0.64, 
0.80, 1.0, 1.25, and 1.56 p,g streptomycin base/mL. Ref. concn 
is 1.0 (xg/mL. 

B. Plates 

(a) Base layer. — Add 12 mL melted agar medium E to ster- 
ile petri dishes. Distribute agar evenly and let harden on per- 
fectly level surface. 

(b) Seed layer. — Before assay, det. by prepn of trial plates 
optimum concn (usually 0.05-0.2%) of spore suspension of 

B. subtilis, 957.23D(d), to be added to agar medium E. For 
actual assay, sharp zones of inhibition (14 mm ± 10%) should 
be obtained with 0.64 jxg streptomycin base/mL. Add appro- 
priate amt of spore suspension to agar medium E which has 
been melted and cooled to 48°. Mix thoroly and add 4.0 mL 
to each plate contg base layer. 

C. Assay Solution 

Obtain and prep, sample as in 965.16 and 950.02. Using 
10.0 g sample and 200 mL 0.5N HC1, shake mech. 30 min or 



blend 2 min in high-speed blender. Centrf. ca 15 min at 2000 
rpm. Transfer aliquot of supernate to beaker, add ca 25 mL 
pH 8 buffer, 957.238(b), and adjust to pH 8 ± 0.1 with 5N 
and IN NaOH. Transfer quant, to suitable vol. flask, dil. to 
vol. with pH 8 buffer, 957.23B(b), and mix. Dil. aliquot with 
enough pH 8 phosphate buffer to obtain estd concn of 1.0 fxg 
streptomycin base/mL. Filter dild soln without suction thru 
Whatman No. 2V paper, or equiv. Designate as assay soln. 

D. Assay 

Using streptomycin std response line, assay soln, and plates, 
proceed as in 957.23E-F, incubating at 37°. 

Refs.: JAOAC 54, 116(1971); 55, 714(1972). 

CAS-57-92-1 (streptomycin) 



972.57 Streptomycin in Feeds 

Microbiological Method 

First Action 1972 
Final Action 1973 

Method II 

(Applicable to feeds contg 5-30 g/ton) 

A. Standard Solutions 

Std response line. — Dil. aliquots of stock soln, 971.49A(a), 
with enough pH 8 buffer, 957*23B(b), to obtain concns of 
0.19, 0.24, 0.30, 0.38, and 0.47 fxg streptomycin base/mL. 
Ref. concn is 0.30 (xg/mL. 

& Plates 

(a) Base layer. — Prep, as in 971.49B(a), except use 10 mL 
agar medium E. 

(b) Seed layer. — Prep, as in 971.49B(b), except that sharp 
zones of inhibition (11 mm ± 10%) should be obtained with 
0.19 (Jtg/mL std. 

C. Assay Solution 

Proceed as in 971. 49C, using 40 g sample and dilg to estd 
final concn of 0.3 fxg streptomycin base/mL. Let soln stand 
1 hr before filtering. 

Note: Feeds may produce considerable gas during acid extn. 
When using mech. shaker, let each sample stand in acid ex- 
tractant ca 1 hr with occasional swirling before placing on 
shaker. When using high-speed blender, use jar with lid cover 
rather than sealed jar, and hold lid down by hand at start of 
blending. 

D. Assay 

Using streptomycin std response line, assay soln, and plates, 
proceed as in 957.23E-F, incubating at 22-25°. 

Refs.: JAOAC 54, 116(1971); 55, 714(1972). 

CAS-57-92-1 (streptomycin) 



962.26 



Tylosin in Feeds 

Microbiological Method 

First Action 1962 
Final Action 1965 



g/ton) 



(Applicable to feeds containing 

A. Standard Solutions 

(a) Tylosin stock soln. — Dry Tylosin base Ref. Std (avail- 
able from Elanco Products Co.) 4 hr at 70° and store in des- 



AOAC Official Methods of Analysis (1990) 



Bacitracin 



129 



iccator over fresh P 2 5 . Accurately weigh suitable amt (10- 
15 mg) of dried std and dissolve in 5 mL MeOH. Adjust vol. 
with pH 7 phosphate buffer, 957.236(c), to give concn of 1000 
|xg/mL. Store in refrigerator <2 weeks. 

(b) Std response line. — 'Oil. appropriate aliquots of stock 
soln with mixt. of MeOH and pH 8 phosphate buffer, 
957.23B(b), (4 + 6), to obtain concns of 0.125, 0.25, 0.50, 
1.0, and 2.0 (xg/mL. Ref. concn is 0.50 |xg/mL.' 

B. Plates 

(a) Base layer. — Add 10 mL melted agar medium H to petri 
dishes, distribute evenly, and let harden on perfectly level sur- 
face. 

(b) Seed layer. — Before assay, det. by prepn of trial plates 
optimum concn of organism suspension of S. lutea (usually 
0.05-0.2% of suspension prepd as in 957.23D(e)(7) or 0.5- 
2% as in 957.23D(e)(2)) to be added to agar medium H to 
obtain zones of inhibition of adequate size (15 mm ± 10% 
with 0.50 (xg/mL) and sharpness. For actual assay, add ap- 
propriate amt of organism suspension to agar medium H melted 
and cooled to 48°. Mix thoroly and add 5.0 mL to each plate 
contg base layer. Refrigerate plates until just before applica- 
tion of assay solns. 

C. Assay Solution 

Obtain and prep, sample as in 965-16 and 950.02. Accu- 
rately weigh 10 g feed premix or 20 g final feed into 250 mL 
homogenizer cup or blender jar. Add 90 mL hot (70-80°) pH 
8 phosphate buffer, 957.23B(b), and place on steam bath 10 
min. Blend 5 min, add 60 mL MeOH, and blend addnl 5 min. 
Centrf. or filter thru Whatman No. 1 paper and dil., if nec- 
essary, with mixt. of MeOH and pH 8 phosphate buffer, 
957.238(b), (4 + 6), to concn of 0.5 \xg tylosin/mL. 

D. Assay 

Prep. 10 plates for std response line and 5 for each sample. 
Place 5 cylinders on each std response line plate at 72° inter- 
vals on 2.8 cm radius. Place 4 cylinders on each sample plate 
at 90° intervals. Fill cylinders on each of 10 std plates with 
each concn of std response line. On each sample plate fill 2 
diagonally opposite cylinders with ref. concn and remaining 2 
cylinders with assay soln. Incubate plates overnight at 30°. 
Measure zones of inhibition to nearest 0.1 mm. Record av. 
zone diam. for each concn of std on std plates and proceed as 
in 957.23E. 

Average the 10 readings of ref. concn on sample plates and 
the 10 readings of assay soln. Proceed as in 957. 23F. 

Ref.: JAOAC45, 317(1962). 

CAS-1401-69-0 (tylosin) 



CHEMICAL METHODS FOR ANTIBIOTICS 

982.44 Bacitracin in Premix Feeds 

Liquid Chromatographic Method 
First Action 1982 

A. Principle 

Bacitracin is extd from feed into acidified org. solv. system. 
Ext is centrfgd, and supernate is analyzed by ion-suppressed 
reverse phase LC with photometric detection at 254 nm. 

B. Reagents and Apparatus 

(a) Liquid chromatography — Hewlett-Packard Model 1084- 
A, equipped with UV photometric detector. Operating con- 
dition: flow rate 2.0 mL/min; detector wavelength 254 nm; 



20 jxL loop injection valve (Valco Instruments Co., Inc., PO 
Box 55603, Houston, TX 77255); ambient temperature. 

(b) Chromatographic column. — 15 cm X 4.6 mm id, con- 
taining 5 |xm Supelcosil LC-8 reverse phase packing. Use col- 
umn for bacitracin analysis only. 

(c) Phosphate-EDTA buffer.— pB. 4.5. Dissolve 13.6 g 
KH 2 P0 4 and 2.5 g EDTA in 1 L H 2 0. 

(d) Phosphate buffer. —pH 6.0. Dissolve 1.5 g K 2 HP0 4 and 
8.5 g KH 2 P0 4 in 1 L H 2 0. 

(e) Solvent systems. — Measure vol. indicated below with 
graduate (except where noted otherwise) into 100 mL vol. flask 
and dil. to vol. with H 2 0: 



Solvent 


A Solv. 


Vol.% 
B. Solv. 


Extg Solv. 


CH 3 CN 
MeOH 

Phosphate-EDTA buffer 
Phosphate buffer 3 
Coned phosphoric acid 3 




20 




40 

12 

20 






28 

28 



3 

1.2 



a Use vol. pipet 

(f) Mobile phase.— Mix 59% (v/v) B solv. with 41% (v/ 
v) A solv. Mix, and adjust pH to 6.8 with NaOH. Slight ad- 
justment to % vol. of B solv. may be required to obtain desired 
sepn. 

C. Preparation of Standard 

(a) Drying of std. — Caution: Bacitracin is very hydro- 
scopic. Dry std day before use and store in desiccator over- 
night. Accurately weigh 130-140 mg bacitracin ref. std (IMC, 
Pitman-Moore Inc., 1331 First St, PO Box 207, Terre Haute, 
IN 47808; 56.3 units/mg) into tared (= A) 50 mL vol. flask. 
Dry std 3 h at 60° under vac. at <5 mm pressure. Remove 
from oven and place in desiccator to cool. Reweigh (- B). 
Amt bacitracin std = B — A. 

(b) Preparation of std soln. — Note: Store stds under refrig- 
eration if not analyzed within 3 h of prepn. Preferably, prep, 
std, store in refrigerator >30 min before analysis, and remove 
from refrigerator just before analysis. Std soln I: Dissolve bac- 
itracin std in 50 mL vol. flask with ca 20 mL extg solv. and 
dil. to vol. Prep, following dilns from this soln. Std soln 2: 
Pipet 20 mL std soln 1 into 25 mL vol. flask; dil. to vol. with 
extg solv. Std soln 3: Pipet 15 mL std soln 1 into 25 mL vol. 
flask; dil. to vol. with extg solv. 

D. Extraction 

Accurately weigh amt of feed contg ca 6000 units bacitracin 
activity into 125 mL erlenmeyer. Add 50 mL extg solv. with 
vol. pipet and ext with wrist-action shaking >5 min. Centri- 
fuge 10 mL portion of ext 2-3 min at 2000-3000 rpm. Use 
clear supernate for assay. Note: Store extd sample soln under 
refrigeration if not analyzed within 3 h. Preferably, prep, sam- 
ple solns, store in refrigerator >30 min before analysis, and 
remove from refrigerator just before analysis. 

E. Determination 

Inject clear supernate from centrfgd feed and std solns into 
chromatograph, starting with std soln, then 2 sample solns, 
and then another std soln, until all samples and stds have been 
injected. Measure and total peak hts of the 3 active component 
peaks (Fig. 982.44) for sample (PH) and std (PH') solns. 

Calc. response line for stds, using least squares linear fitting 
of following equation: 

PH* = w(P') + b 

where PH' = peak hts of std solns 1 , 2, and 3; P' — potency 
of std soln in units/50 mL for std solns 1, 2, and 3; m, b =.. 
least squares detd slope and intercept. 



130 



Drugs in Feeds 



AOAC Official Methods of Analysis (1 990) 




= TIME 

FIG. 982.44— Liquid chromatogram of bacitracin active com- 
ponents (indicated by arrows) 



Det. bacitracin content of feed from: 

0.01080 (PH - b) 

g bacitracin /lb = 

m x sample wt 

where 0.01080 = 453.6 (g/lb)/42 000 (units/g bacitracin). 

Ref.: JAOAC65, 1178(1982). 

CAS- 1405-87-4 (bacitracin) 



968.49 Chlortetracycline in Feeds 

Microscopic Test 

First Action 1968 
Final Action 1969 

A. Apparatus 

Microscopes.— See 964.07A(a) and (b). 

B. Reagent 

Modified Sakaguchi reagent. — Dissolve 5 g H3BO3 in 150 
mL H 2 and add 350 mL H 2 S0 4 . Store in g-s bottle in re- 
frigerator. Use cold. 

C. Determination 

Grind sample to pass sieve with circular openings 1 mm (V25 
in.) diam. and mix thoroly. If sample cannot be ground, re- 
duce to as fine condition as possible. Do not grind molasses 
feeds. Pipet ca 10 mL Sakaguchi reagent into 9 cm petri dish. 
Place No. 60 sieve over petri dish. With top of spatula, sprin- 
kle ca 0.5 g sample on sieve, and gently tap it to obtain good 
distribution of particles over liq. surface. Place under stereo- 
scopic microscope and examine with transmitted light at ca 



15 X . If substage illumination is not available, place petri dish 
on white surface and illuminate with blue light. 

As particles of antibiotic slowly dissolve, diffusing chlor- 
tetracycline turns intense purple. Color fades in 5-10 min. 

Refs.: JAOAC 51, 750(1968); 59, 357(1976). 

CAS-57-62-5 (chlortetracycline) 



966.30 Griseofulvin in Feeds 

Spectrophotometric Method 

First Action 1966 
Final Action 1975 

(Applicable to concns >10 mg/oz.) 

{Caution: See safety notes on distillation, toxic solvents, and 
chloroform.) 

A. Reagents 

(a) Activated alumina. — Alcoa grade F-20. 

(b) Solvent mixture.— Pet ether-CHCl 3 (65 + 35). 

(c) Griseofulvin std soln. — 10 |mg/mL. Accurately weigh 
ca 25 mg USP Griseofulvin Ref. Std into 250 mL vol. flask; 
dissolve and dil. to vol. with solv. mixt. Dil. 10 mL of this 
soln to 100 mL in vol. flask. 

B. Apparatus 

(a) Chromatographic tube. — 20 X 400 mm, with fritted 
disk and stopcock. 

(b) High-speed blender. — Waring-type, or equiv., 1 L 
capacity. 

(c) Spectrophotometer. — Capable of accurate readings at 
290 and 320 nm. 

C. Preparation of Sample 

Grind 250 g feed pellets or mash in high-speed blender 5 
min. Accurately weigh ca 14 g finely powd feed into fat-free 
thimble and ext in Soxhlet app. 2 hr with 100 mL CHC1 3 . 
Evap. ext to 10 mL on steam bath, dil. with 100 mL pet ether, 
and chromatograph. 

D. Preparation of Chromatographic Column 

Place 50 mL solv. mixt., (b), in tube and add 45 mL 
activated alumina portionwise, with tapping to ensure uniform 
packing. Place small glass wool pad on top of alumina and 
drain solv. to just below top of pad. 

E. Determination 

Add pet ether-CHCl 3 sample ext to column. As last of ext 
passes thru glass wool pad, rinse sample flask with solv. mixt., 
add to column, and begin elution with solv. mixt. Adjust liq. 
head to give flow rate of 15-20 mL/min. Start collecting 25 
mL fractions when green eluate first appears (discard yellow 
and almost colorless eluates which precede). When A of frac- 
tions at 290 nm exceeds A at 320 nm, stop fractionating, and 
collect next 700 mL eluate. Dil. eluate to vol. in 1 L vol. flask 
with solv. mixt. Det. A of this soln and of griseofulvin std 
soln at 290 and 320 nm against solv. mixt. blank. 

mg Griseofulvin/oz = (A? 90 — A 32 o)(W) 

(10) (28.35)/(A 290 - ^20) (25) (g sample) 



where A refers to sample eluate, A 1 to std soln, and W 
ref. std griseofulvin used to prep, std soln. 



mg 



AOAC Official Methods of Analysis (1990) 



Lasalocid 



131 



Ref.: JAOAC 49, 494(1966). 
CAS- 126-07-8 (griseofulvin) 



975.61 Lasalocid in Feeds 

Spectrofluorometric Method 

First Action 1975 
Final Action 1977 

A. Principle 

Compd is extd from pH 4.7 soln with EtOAc, fluorescent 
impurities are removed by acid and alkali treatments, and compd 
is detd fluorometrically, correcting for nonspecific fluores- 
cence by complexing with H.3BO3. Monensin and ethoxyquin 
do not interfere. 

B. Apparatus 

Spectrofluorometer . — With 10 mm fused quartz cells. 
Excitation and emission wavelengths, ca 310 and 419 nm, resp. 
Accurately det. peak excitation and emission wavelengths us- 
ing std soln I, following manufacturer's directions. Do not 
change wavelength settings between readings. 

For routine setting and checking of instrument, use std 
soln I. 

Adjust settings to compensate for decreased intensity with 
age from dulled reflecting surfaces and lamp. 

C. Reagents 

(a) Acetate buffer soln. — pH 4.7. Dissolve 5.0 g NaOAc 
in ca 50 mL H 2 0, adjust to pH 4.7 with HO Ac, and dil. to 
100 mL with H 2 0. 

(b) Ethyl acetate. — Must have ca fluorescence. If nec- 
essary, purify as follows: Elute 1 gal. EtOAc thru 9-10 cm 
(od) column packed with ca 100 cm silica gel (activated des- 
iccant, 100-200 mesh, Grade H, W. R. Grace & Co., Davison 
Chemical Div., 10 E. Baltimore St, PO Box 2117, Baltimore, 
MD) topped with 5 cm layer of NaHCO> Redistil eluate from 
all-glass app. with 60 cm jacketed distg column, discarding 
first and last 10%. {Caution: See safety notes on distillation 
and ethyl acetate.) To redistd EtOAc add 40% aq. NaOH and 
mix briefly. Follow with anhyd. Na 2 S0 4 and shake again. (Ra- 
tio of EtOAc:40% NaOH:Na 2 S0 4 is 1000:50:200 or multiple 
thereof.) 

(c) Methanolic boric acid soln. — Dissolve 20.0 g H 3 B0 3 
in MeOH and dil. to 500 mL with MeOH. Prep, fresh daily. 

(d) Lasalocid std solns. — (1) Std soln I. — Dissolve 30.0 
mg Lasalocid Ref. Std (available from Hoffmann-La Roche 
Inc.) in EtOAc and dil. to 100 mL with EtOAc. Pipet 4 mL 
into 100 mL vol. flask, dil. to vol. with EtOAc, and mix. Pipet 
25 mL final diln into 50 mL g-s centrf. tube contg 2.4 mL pH 
4.7 buffer. Shake mech. 25-30 min and centrf. 10 min at 2000 
rpm. Pipet 2 mL clear EtOAc ext into 100 mL vol. flask, dil. 
to vol. with EtOAc, and mix. (2) Std soln II. — Pipet addnl 2 
mL clear EtOAc ext into another 100 mL vol. flask contg 10 
mL methanolic H 3 B0 3 soln, dil. to vol. with EtOAc, and mix. 

D. Preparation of Sampie 

(a) Feeds. — Grind 200 g sample (mash, pellets, or crum- 
bles) to pass No. 30 sieve, and mix thoroly. Accurately weigh 
ca 4 g sample into 50 mL g-s centrf. tube contg 2.4 mL pH 
4.7 buffer. Turn and shake tube by hand to wet uniformly. 
Immerse tube 4-5 min in 70° H 2 bath. Cool to room temp, 
and add 25 mL EtOAc by pipet. Stopper tube and shake briefly 
but vigorously by hand to disperse sample. If necessary, break 



up lumps with narrow-tip spatula or glass rod. Stopper tube 
and shake mech. 25-30 min. Centrf. 10 min at 2000 rpm. 
Pipet 15 mL clear EtOAc ext into 50 mL centrf. tube. Add 2 
mL L5/V HC1 and shake 10 min. Centrf. 10 min at 2000 rpm. 
Pipet 10 mL clear EtOAc ext into another 50 mL g-s centrf. 
tube and add 0.5 mL 40% NaOH soln. Shake briefly by hand, 
add 2 g anhyd, Na 2 S0 4 , and shake again. Centrf. 10 min at 
2000 rpm. If EtOAc soln is not clear or fine particles are pres- 
ent at surface, swirl tube gently by hand and recentrf. Pipet 2 
mL EtOAc layer into 100 mL vol. flask without disturbing aq. 
alk. soln, dil. to vol. with EtOAc, and mix. Designate as Sam- 
ple soln I. Pipet another 2 mL aliquot EtOAc layer into second 
100 mL vol. flask contg 10 mL methanolic H 3 B0 3 soln, dil. 
to vol. with EtOAc, and mix. Designate as Sample soln II. 

Pipet 25 mL EtOAc into 50 mL g-s centrf. tube contg 2.4 
mL pH 4.7 buffer and proceed as above, beginning "Stopper 
tube and shake briefly ..." Designate final solns as Reagent 
blank soln I and Reagent blank soln II. 

(b) Premixes. — Accurately weigh 2.00 g 15% premix and 
transfer into 500 mL vol. flask. Add exactly 250 mL EtOAc 
and shake 25 min on mech. shaker. Centrf. aliquot, and dil. 
with EtOAc as in (a) to obtain Sample soln I and Sample soln 
II (complex) contg ca 0.24 u.g lasalocid/mL EtOAc. Proceed 
with fluorescence measurements as in 975. 61E. (Note: Omit 
treatment with pH 4.7 buffer for both premix and std.) 

E. Determination 

Set excitation and emission wavelengths of app. at max. 
Adjust instrument with std. soln I, (d)(/), in cell to microam- 
meter reading of 0.400. Check this ref. point before and after 
each reading, using same cell for all ref. readings. Because of 
decomposition in UV, discard and replace std soln I after every 
second reading. Measure fluorescence at 419 nm in order: 
Sample soln I (U\), Std soln I (Si), Reagent blank soln I (R\), 
Sample soln II (U 2 ), Std soln II (5 2 ), and Reagent blank soln 
II (R 2 ). If reading of std soln I drifts, adjust gain to initial 
setting. If drift is beyond 0.393-0.407, recheck readings of all 
solns. 

Altho fluorescence response of std is linear from 0.12 to 
0.48 |ag/mL, concn of lasalocid in sample soln should be ± 
25% of that of std soln. 

F. Calculations 

(a) % Lasalocid Na in feed = [(17, - Rj) 

- (U 2 - R 2 )} x D x S/[(S, - R } ) - (S 2 - R 2 )} 

x W x 96 x 100 

where V , S u S 2 , andtf are defined in 975. 61E; D - diln factor 
(25 x 100/2 = 1250); S = concn of lasalocid in Std soln I ( = 
0.24 |xg/mL); W = g sample; and 96 = % recovery. When S 
- 0.24 |xg/mL, W = 4.00 g, and tf , and R 2 = 0, 

% Lasalocid Na in feed - (U t - U 2 ) X 0.00781/(5, -5 2 ) 

(b) % Lasalocid Na in premix - \(U , - R f ) ~ (U 2 - R 2 )\ 

x D x Si\(S } - Ri) - (S 2 - R 2 )] x W x 10,000 

where V, S U S 2 , and R are defined in 975.61E; D = diln factor 
(250 x 50 x 50 x 100/5 x 5 x 2 = 1,250,000); S = concn 
of lasalocid in Std soln I (= 0.24 |xg/mL); W - g sample. 
When S = 0.24 |ULg/mL, W = 2.00 g and /?, and R 2 = 0, 

% Lasalocid Na in 15% premix 

- (U } - U 2 ) x 15/(5, - S 2 ) 

Ref.: JAOAC 58, 507(1975). 

CAS-25999-31-9 (lasalocid) 



132 



Drugs in Feeds 



AOAC Official Methods of Analysis (1990) 



Common and Chemical Names of Drugs in this Chapter 



Common Name 



Chemical Name 



Aklomide 
Amprolium 
Arprinocid 
Arsanilic acid 
Bithionol 
Buquinolate 
Carbadox 
Chlortetracycline 
hydrochloride 
Decoquinate 
Dibutyltin diiaurate 
Diethylstilbestrol 
Dimetridazole 
Ethopabate 
Furazolidone 
Glycarbylamide 
Griseofulvin 
Hygromycin B 

Ipronidazole 
Lasalocid 

Lincomycin 

Melengestrol (acetate) 

Nequinate 

Nicarbazin 

Nicotine 

Nifursoi 

Nihydrazone 

Nitarsone 

Nithiazide 

Nitrodan 

Nitrofurazone 

Nitromide 

Nitrophenide 

Oxy tetracycline 

Penicillin G procaine 

Phenothiazine 

Pyrantel tartrate 

Racephenicol 

Reserpine 

Ronnel 

Roxarsone 

Spectinomycin 

Streptomycin 

Sulfadimethoxine 

Sulfaguanidine 

Sulfamerazine 

Sulfamethazine 

Sulfanitran 

Sulfaquinoxaline 

Sulfathiazole 

Thiabendazole 

Zoalene 



2-Chloro-4-nitrobenzamide 

1-[(4-Amino-2-propyl-5-pyrimidinyl)methyl]-2-methylpyridinium chloride 

9-[2-Chloro-6-fluorophenyl)methy|]-9H-purin-6-amine 

(4-Aminophenyl)arsonic acid 

2,2'-Thiobis(4,6-dichlorophenol) 

4-Hydroxy-6,7-bis(2-methylpropoxy)-3-quinolinecarboxylic acid ethyl ester 

2-(2-Quinoxalinylmethy)ene)hydrazinecarboxylic acid methyl ester N\/V 4 -dioxide 

(4a,4aa,5aa,60,1 2aa)-7-Chloro-4-(dimethylamino)-1 ,4,43,5,53,6,1 1 ,1 2a-octahydro-3,6,10,12,1 2a-pentahydroxy-6-methyl-1 ,1 1 -dioxo- 

2-naphthacenecarboxamide monohydrochloride 
6-(Decyloxy)-7-ethoxy-4-hydroxy-3-quinolinecarboxyiic acid ethyl ester 
Dibutylbis[(1-oxododecyl)oxy]stannane 
4,4'-(1 ,2-Diethyl-1 ,2-ethenediyl)bisphenol 
1 ,2-Dimethyl-5-nitro-1 H-imidazole 
4-(Acetamido)-2-ethoxybenzoic acid methyl ester 
3-[[(5-Nitro-2-furanyl)methylene]amino]-2-oxazolidinone 
1 H-lmidazole-4,5-dicarboxamide 

7-Chloro-2',4,6-trimethoxy-6'-methyl-spiro[benzofuran-2(3H),1'-[2]cyclohexene]-3,4'-dione 
C^6-Amino-6-deoxy-L-glycero-D-galacto-heptopyranosylidene-(1~^2-3)-0-p-D-talopyranosyl-(1-^5)-2-deoxy-/V 3 -methyl- 

D-streptamine 
1-Methyl-2-(1-methylethyl)-5-nitro-1H-imdazole 
6-[7(/?)-[5(S)-Ethyl-5-(5{fl)-ethyltetrahy 

4-(S)-hydroxy-3-(R),5(S)-dimethyl-6-oxononyl]-2-hydroxy-3-methylbenzoic acid 
Methyl-6,8-dideoxy-6[[1-methyl-4-propyl-2-pyrrolidinyl)carbonyl]amino)-1-thio-D-ery^ro-a-D-gia/acto-octopyranoside 
1 7-Hydroxy-6-methyl-1 6-methylenepregna- 4,6-diene-3,20-dione 
6-Butyl-1,4-dihydro-4-oxo-7-(phenylmethoxy)-3-quinolinecarboxylic acid 
N,/V'-Bis(4-nitrophenyl)urea compd with 4,6-dimethyt-2(1H)-pyrimidinone (1:1) 
3-(1-Methyl-2-pyrrolidinyl)pyridine 

[(5-Nitro-2-furanyl)methylene]hydrazide-2-hydroxy-3,5-dinitrobenzoic acid 
5-Nitrofurfurylidenehydrazide acetic acid 
(4-Nitrophenyl)-arsonic acid 
W-Ethyl-A/'-(5-nitro-2-thiazolyl)urea 
3-Methyl-5-[(4-nitrophenyi)azo]-2-thioxo-4-thiazolidinone 
2-[(5-Nitro-2-furanyl)methylene]hydrazinecarboxamide 
3,5-Dinitrobenzamide 
Bis(m-nitrophenyi)disulfide 
(4a,4aa,5a,5aa,63,12aa)-4-(Dimethylamino)-1,4,4a,5,5a ! 6,11,12a-octahydro-3,5,6,10,12,12a-hexahydroxy-6-methyl-1,11-dioxo-2- 

naphthacenecarboxamide dihydrate 
(2tt,5a,6p)-3,3-Dimethyl-7-oxo-6-[(phenylacetyl)amino]-4-thia-1-azabicyc!o[3.2.0]heptane-2-carboxylic acid 
Thiodiphenylamine 

E-1,4,5,6-Tetrahydro-1-methyl-2-[2-(2-thienyl)vin]pyrimidine tartrate (1:1) 
2,2-Dichloro-A/-[2-hydroxy-1-(hydroxymethyl)-2~[4-(methylsulfonyl)phenyl]ethyl]-acetamide 

(3p,16p,17a,18|3,20a)-1 1-17-Dimethoxy-18-[(3,4,5-trimethoxybenzoyl)oxy]yohimban-16-carboxylic acid methyl ester 
0,0-Dimethyl 0-(2,4,5-trichlorophenyl) phosphorothioic acid ester 
(4-Hydroxy-3-nitrophenyl)-arsonic acid 

Decahydro^a^.g-trihydroxy^-methyl-e^-bistmethylaminoJ^H-pyrano^.S-oKl^lbenzodioxin^-one 
0-2-Deoxy-2-(methylamino)-a-L"glucopyranosyl-(1^2)-05-deoxy-3-C-formyl-a-L-lyxofuranosyl-(1-^4)-A/ J A/'-bis(aminoiminomethyl)- 

D-streptamine 
4-Amino-A/-(2,6-dimethoxy-4-pyrimidinyl)-benzenesulfonamide 
4-Amino-/V-(diaminomethy!ene)-benzenesulfonamide 
4-Amino-/V-(4-methyl-2-pyrimidinyl)-benzenesulfonamide 
4-Amino-A/-(4,6-dimethyl-2-pyrimidinyl)-benzenesulfonamide 
N-[4-[[(4-Nitrophenyl)amino]sulfonyl]phenyl]-acetamide 
4-Amino-A/-2-quinoxalinyl-benzenesulfonamide 
4-Amino-/V-2-thiazolyl-benzenesulfonamide 
2-(4-Thiazolyl)-1H-benzimidazole 
2-Methyl-3,5-dinitrobenzamide 



Sources: U SAN and the (JSP Dictionary of Drug Names (1989). U.S. Pharmacopeia! Convention, Rockville, MD; The Merck index (1989) 10th ed., 
Co., Inc., Rahway, NJ. 



Merck & 



6 B Disinfectants 

Aram BeSosan, Associate Chapter Editor 

Environmental Protection Agency 



PHENOL COEFFICIENT METHODS 

955.11 Testing Disinfectants 

against Salmonella typhi 

Phenol Coefficient Method 
Final Action 1964 

(Applicable to testing disinfectants miscible with H 2 that do 

not exert bacteriostatic effects that cannot be neutzd by one of 

subculture media specified, or overcome by suitable subtrans- 

fer procedures. The 95% confidence limits are ±12%.) 

A. Culture Media 

(a) Nutrient broth. — Boil 5 g beef ext (Difco), 5 g NaCl, 
and 10 g peptone (Anatone, peptic hydrolysate of pork tissues, 
manufactured by American Laboratories, Inc., 4410 S 102nd 
St, Omaha, NE 68127) in 1 L H 2 20 min, and dil. to vol. 
with H 2 0; adjust to pH 6.8. (If colorimetric method is used, 
adjust broth to give dark green with bromothymol blue.) Filter 
thru paper, place 10 mL portions in 20 x 150 mm test tubes, 
and autoclave 20 min at 121°. Use this broth for daily transfers 
of test cultures. 

(b) Synthetic broth. — Soln A: Dissolve 0.05 g L-cystine, 0.37 
g DL-methionine, 0.4 g L-arginine.HCl, 0.3 g DL-histi- 
dine.HCI, 0.85 g t-lysine.HCl, 0.21 g L-tyrosine, 0.5 g dl- 
threonine, 1 .0 g dl- valine, 0.8 g L-leucine, 0.44 g DL-isoleu- 
cine, 0.06 g glycine, 0.61 g DL-serine, 0.43 g DL-alanine, 1 .3 
g L-glutamic acid. HO, 0.45 g L-aspartic acid, 0.26 g DL-phen- 
ylalanine, 0.05 g dl- tryptophan, and 0.05 g l- proline in 500 
mL H 2 contg 18 mL IN NaOH. 

Soln B: Dissolve 3.0 g NaCl, 0.2 g KC1, 0. 1 g MgS0 4 .7H 2 0, 
1.5 g KH 2 P0 4 , 4.0 g Na 2 HP0 4 , 0.01 g thiamine.HCl, and 0.01 
g niacinamide in 500 mL H 2 0. 

Mix Solns A and B, dispense in 10 mL portions in 20 X 
150 mm tubes, and autoclave 20 min at 121°. Before using 
for daily transfers of test cultures, aseptically add 0. 1 mL ster- 
ile 1.0% glucose soln per tube. Grow cultures with tube slanted 
8° from horizontal. 

(c) Nutrient agar. — Dissolve 1.5% Bacto agar (Difco) in 
nutrient broth and adjust to pH 1 .2-1 A (blue-green with 
bromothymol blue) or in synthetic broth, tube, autoclave, and 
slant, 

(d) Subculture media. — Use (/), (2), or (3), whichever gives 
lowest result. (Com. dehydrated brands made to conform with 
preceding specifications may be used.) With oxidizing prod- 
ucts and products formulated with toxic compds contg certain 
heavy metals like Hg, (2) will usually give lowest result. With 
products contg cationic surface active materials, (3) will usu- 
ally give lowest result. See also 955. 14C, par. 5 

(J) Nutrient broth described in (a). 

(2) Fluid thioglycolate medium USP XX: Mix 0.5 g L-cys- 
tine, 0.75 g agar, 2.5 g NaCl, 5.5 g glucose. H 2 0, 5.0 g H 2 0- 
sol. yeast ext, and 15.0 g pancreatic digest of casein with 1 
L H 2 0. Heat on H 2 bath to dissolve, add 0.5 g Na thiogly- 
colate or 0.3 g thioglycolic acid, and adjust with 17V NaOH to 
pH 7.1 ± 0.2. If filtration is necessary, reheat without boiling 
and filter hot thru moistened filter paper. Add 1.0 mL freshly 



prepd 0.1% Na resazurin soln, transfer 10 mL portions to 20 
X 150 mm tubes, and autoclave 20 min at 121°. Cool at once 
to 25° and store at 20-30°, protected from light. 

(3) "Letheen broth" : Dissolve 0.7 g lecithin (Asolectin, As- 
sociated Concentrates, 32-34 61st St, Woodside, NY 11377) 
and 5.0 g polysorbate 80 (Tween 80, or equiv.) in 400 mL 
hot H 2 and boil until clear. Add 600 mL soln of 5.0 g beef 
ext (Difco), 10.0 g peptone (Anatone, (a)), and 5 g NaCl in 
H 2 0, and boil 10 min. Adjust with IN NaOH and /or IN HO 
to pH 7.0 ± 0.2 and filter thru coarse paper; transfer 10 mL 
portions to 20 x 150 mm tubes, and autoclave 20 min at 121°. 

(4) Cystine trypticase agar (BBL Microbiology Systems): 
Suspend 29.5 g in 1 L H 2 0. Heat gently with frequent agi- 
tation and boil ca 1 min or until soln is complete. Transfer 10 
mL portions to 20 x 150 mm tubes, and autoclave 15 min at 
12 lb pressure. Cool in upright position and store <25 days at 
20-30°. Use for monthly transfer of stab stock cultures of Ps. 
aeruginosa PRD 10 (ATCC 15442). 

(5) Other subculture media: Use (d)(2) with 0.7 g lecithin 
(Asolectin, Associated Concentrates, Inc.) and 5.0 g polysor- 
bate 80 (Tween 80, or equiv.) added; or suspend 29.8 g prepd 
fluid thioglycolate medium (Difco), 0.7 g lecithin, and 5.0 g 
polysorbate 80 in 1 L H 2 0, and boil until soln is clear. Cool, 
dispense in 10 mL portions in 20 x 150 mm tubes, and au- 
toclave 20 min at 121°. Store at 20-30°. Protect from light. 

B. Apparatus and Reagents 

(a) Glassware. — 1, 5, and 10 mL vol. pipets; 1, 5, and 10 
mL Mohr pipets graduated to 0. 1 mL or less; 100 mL g-s cyl- 
inders graduated in 1 mL divisions; Pyrex lipped test tubes, 
25 x 150 mm (medication tubes) reusable or disposable bo- 
rosilicate; bacteriological culture tubes, 20 x 150 mm (test 
culture and subculture tubes). Plug medication tubes with cot- 
ton wrapped in 1 layer of cheesecloth (tubes capped with Mor- 
ton closures are an acceptable alternative). Sterilize all glass- 
ware 2 hr in hot air oven at 180°. Loosely plug pipets with 
cotton at mouth and place in closed metal containers before 
sterilizing. 

(b) Water bath. — Const temp, relatively deep H 2 bath ca- 
pable of maintaining 20 ± 0.2°, with cover having > 10 well- 
spaced holes which admit medication tubes but not their lips. 

(c) Racks. — Any convenient style. Blocks of wood (size 
depending on space in incubator) with deep holes are satis- 
factory. Have holes well spaced to ensure quick manipulation 
of tubes. It is convenient to have them large enough to admit 
medication tubes while dilns are being made. 

(d) Transfer loop. — Make 4 mm id single loop at end of 
50^75 mm (2-3") Pt or Pt alloy wire No. 23 B&S gage or 4 
mm loop fused on 75 mm (3") shaft (available from Matthey- 
Bishop, Inc., 1401 King Rd, West Chester, PA 19380). Fit 
other end in suitable holder (glass or A J rod). Bend loop at 
30° angle with stem, Fig. 955.11. 

(e) Test organism. — Hopkins strain 26 of Salmonella typhi 
(Schroeter) Warren and Scott, ATCC No. 6539 (formerly called 
Bac. typhosus and E be r the I la typhosa). Maintain stock culture 
on nutrient agar slants by monthly transfers. Incubate new stock 
transfer 2 days at 37°; then store at 2-5°. From stock culture 
inoculate tube of nutrient broth and make at least 4 consecutive 



133 



134 



Disinfectants 



AOAC Official Methods of Analysis (1990) 




FIG. 955.11 — Transfer loop and manner of using in phenol 
coefficient technic 



daily transfers (^30) in nutrient broth, incubating at 37°, be- 
fore using culture for testing. (If only 1 daily transfer has been 
missed, it is not necessary to repeat the 4 consecutive trans- 
fers.) Use 22-26 hr culture of organism grown in nutrient broth 
at 37° in test. Shake, and let settle 15 min before using. 
With Ps. aeruginosa PRD 10, proceed as in 964.02. 

(f) Phenol stock soln. — 5% (w/v). Weigh 50 g USP phenol, 
which congeals at >40°, in beaker. Dissolve in H 2 0, rinse soln 
into 1 L vol. flask, and dil. to vol. Stdze with 0.17V KBr- 
KBr0 3 soln, (g), as follows: Transfer 25 mL stock soln to 500 
mL vol. flask and dil. to vol. with H 2 0. Transfer 15 mL ali- 
quot of dild soln to 500 mL 1 flask and add 30 mL std KBr- 
KBr0 3 soln. Add 5 mL HO and immediately insert stopper. 
Shake frequently during 30 min and let stand 15 min. Remove 
stopper just enough to quickly add 5 mL 20% KI soln, taking 
care that no Br vapors escape, and immediately stopper flask. 
Shake thoroly, remove stopper, and rinse it and neck of flask 
with little H 2 so that washings flow into flask. Titr. with 
0.1/V Na 2 S 2 3 , using starch indicator: Mix ca 2 g finely powd. 
potato starch with cold H 2 to thin paste; add ca 200 mL boil- 
ing H 2 0, stirring constantly, and immediately discontinue 
heating. Add ca 1 mL Hg, shake, and let stand over the Hg. 
1 mL 0. 1/V KBr-KBr0 3 = 0.001569 g phenol. 

% phenol in stock soln = (30 - mL 0.LV Na 2 S 2 3 

soln from titrn) X 0.001569 x 1333 X 100/1000 

where 30 = mL 0AN KBr-KBr0 3 soln added, 0.001569 - g 
phenol equiv. to 1 mL 0AN KBr-KBr0 3 soln, 1333 = diln 
factor, and 1000 = original vol. phenol stock soln. 

If necessary, adjust stock soln to 5.00 ± 0.05% phenol by 
adding H 2 or phenol. Keep in well stoppered amber bottles 
in cool place, protected from light. 

(g) Potassium hromide-hr ornate soln. — 0.17V. Prep, as in 
947. 13A. Stdze as follows: Transfer 30 mL to I flask, and add 
25 mL H 2 0, 5 mL 20% KI soln, and 5 mL HO. Shake thoroly 
and titr. with O.lvV Na 2 S 2 3 , using starch indicator. 

C. Operating Technic 

Make 1% stock diln of substance to be tested (or any other 
convenient diln, depending on anticipated concn) in g-s cyl- 
inder. Make final dilns, from 1% stock diln, directly into med- 



ication tubes and remove all excess >5 mL. (Range of dilns 
should cover killing limits of disinfectant in 5-15 min and 
should at same time be close enough for accuracy.) From 5% 
stock phenol soln (1-20) dil. further to make 1-90 and 1-1.00 
dilns, and place in medication tubes. Place these tubes, contg 
5 mL each of final dilns of disinfectant and of phenol, and 
tube contg test culture in H 2 bath at 20° and leave 5 min. 
Add 0.5 mL test culture to each of dilns at time intervals cor- 
responding to intervals at which transfers are to be made. (Thus, 
by time 10 tubes have been seeded at 30 sec intervals, 4.5 min 
has elapsed, and 30 sec interval intervenes before transference 
to subculture begins.) Add culture from graduated pipet large 
enough to seed all tubes in any one set. In using Ps. aerugi- 
nosa PRD 10 (ATCC 15442), proceed as in 964.02. 

In inoculating medication tubes, hold them in slanting po- 
sition after removal from bath, insert pipet to just above sur- 
face of disinfectant, and run in culture without letting tip touch 
disinfectant. After adding culture, agitate tubes gently but tho- 
roly to ensure even distribution of bacteria, and replace in bath; 
5 min after seeding first medication tube, transfer 1 loopful of 
mixt. of culture and dild disinfectant from medication tube to 
corresponding subculture tube. To facilitate transfer of uni- 
form drops of medication mixt., hold tube at 60° angle, and 
withdraw loop so that plane of loop is parallel with surface of 
liq. (Fig. 955.11). After 30 sec, transfer loopful from second 
medication tube to second subculture tube and continue pro- 
cess for each successive diln; 5 min after making first transfer, 
begin second set of transfers for 10 min period, and finally 
repeat for 15 min. period. 

Gently agitate medication tubes before taking each interval 
loop subsample for transfer to subculture medium. Before each 
transfer, heat loop to redness in flame and flame mouth of 
every tube. Sterilize loop immediately after each transfer (be- 
fore replugging tubes) to allow time for cooling. Use care in 
transferring and seeding to prevent pipet or needle from touch- 
ing sides or mouth of medication tube, and see that no cotton 
threads adhere to inner sides or mouths of tubes. Incubate sub- 
culture 48 hr at 37° and read results. Thoroly agitate individual 
subculture tubes before incubation. Macroscopic examination 
is usually sufficient. Occasionally 3-day incubation period, agar 
streak, microscopic examination, or agglutination with anti- 
typhoid serum may be necessary to det. feeble growth or sus- 
pected contamination. 

D. Calculation 

Express results in terms of phenol coefficient number, or 
highest diln killing test organism in 10 min but not in 5 min, 
whichever most accurately reflects germicidal value of disin- 
fectant. Phenol coefficient is number obtained by dividing nu- 
merical value of greatest diln (denominator of fraction ex- 
pressing diln) of disinfectant capable of killing S. typhi in 10 
min but not in 5 min by greatest diln of phenol showing same 
results. 

Example: 





Disinfectant (X): 


Diln 


5 Min 10 Min 15 Min 


1-300 





1-325 


-t-0 


1-350 


+ 00 


1-375 


+ +0 


1-400 


+ + + 




Phenol: 


1- 90 


+ 00 


1-100 


+ + + 


350 
Phenol coefficient would be - 3.89 




90 



Test is satisfactory only when phenol control gives one of fol- 
lowing readings: 



AOAC Official Methods of Analysis (1990) 



Use Dilution 



135 



Phenol 


5 Min 


10 Min 


15 Min 


1- 90 


+ or 


+ or 





1-100 


+ 


+ 


+ or 



If none of dilns of disinfectant shows growth in 5 min and 
killing in 10 min, est. hypothetical diln only when any 3 con- 
secutive dilns show following results: first, no growth in 5 
min; second, growth in 5 and 10 min but not in 15 min; and 
third, growth in 5, 10, and 15 min. 



Example: 





Disinfectant (X): 




Diln 


5 Min 




10 Min 


15 Min 


1-300 













1-350 


+ 




+ 





1-400 


+ 


Phenol - 


+ 


+ 


1- 90 













1-100 


+ 




+ 






Phenol coefficient would be - 



325 
95 



^3.42 



To avoid giving impression of fictitious accuracy, calc. phenol 
coefficient to nearest 0.1. Thus, in examples cited above, phenol 
coefficients would be reported as 3.9 and 3.4, instead of 3.89 
and 3.42. 

Note: Although it is commonly accepted criterion that dis- 
infectants be at diln equiv. in germicidal efficiency to phenol 
against S. typhi by calcg 20 x S. typhi coefficient to det. num- 
ber of parts H 2 in which 1 part disinfectant may be mixed, 
this should be regarded as presumptive and is subject to con- 
firmation by use-dilution method. 

Refs.: J. Roy. Sanit. Inst. 24, 424(1903). Am. J. Public Health 
3, 575(1913). U.S. Dept. Agric. Circ. 198 (1931). 
JAOAC 32, 408(1949); 38, 465(1955). Soap Chem. 
Spec. 34, No. 10, 79(1958); 47, 176(1964); 53, 
860(1970); 56, 308(1973). 



955.12 Testing Disinfectants against 

Staphylococcus aureus 
Phenol Coefficient Method 
Final Action 

Proceed as in 955.11, except change phenol dilns and test 
organisms. Use 22-26 hr culture of Staph, aureus FDA 209, 
ATCC No. 6538, having at 20° at least resistance indicated by 
following: 



Phenol 


5 Min 


10 Min 


15 Min 


1-60 


+ or 


+ or 





1-70 


+ 


+ 


+ 



Refs.: J. Roy. Sanit, Inst. 24, 424(1903). Am. J. Public Health 
3, 575(1913). U.S. Dept. Agric. Circ. 198 (1931). 
JAOAC 32, 408(1949); 38, 465(1955). Soap Chem. 
Spec. 34, No. 10, 79(1958). 



955.13 Testing Disinfectants against 

Pseudomonas aeruginosa 
Phenol Coefficient Method 
First Action 

Proceed as in 955.11. Use 22-26 hr culture of Ps. aeru- 
ginosa PRD 10 (ATCC 15442), having resistance to phenol at 
20° at least as follows: 



Phenol 
1-80 
1-90 



5 Min 
+ or 

+ 



10 Min 
+ or 

+ 



15 Min 


+ 



USE-DILUTION METHODS 1 

(Applicable to testing disinfectants miscible with H 2 to 
confirm phenol coefficient results and to det. max. dilns ef- 
fective for practical disinfection. These microbiological meth- 
ods are technique-sensitive methods in which extreme adher- 
ence to the method with identified critical control points, good 
microbiological techniques, and quality controls is required for 
proficiency and validity of results. These methods have been 
validated using distd H 2 only without soil challenge.) 



955.14 Testing Disinfectants against 

Salmonella choleraesuis 

Use-Dilution Method 

First Action 1953 

Final Action 1959 

Repealed First Action 1988 

A. Reagents 

(a) Culture media, — See 955.11 A. 

(b) Test organism, Salmonella choleraesuis. — (ATCC 
10708). Obtain annually directly from ATCC. Maintain stock 
culture on nutrient agar slants by monthly transfers. Incubate 
new stock transfer 2 days at 37°; then store at 2-5°. From stock 
culture inoculate tube of nutrient broth and incubate at 37°. 
Make 3 consecutive 24 hr transfers; then inoculate tubes of 
nutrient broth (2 for each 10 carriers to be tested), using one 
loop of inoculum with each tube; incubate 48-54 hr at 37°. 

(c) Phenol.— See 955.11B(f). 

(d) Sterile distilled water. — Prep, stock supply of H 2 in 
1 L flasks, plug with cotton, sterilize 20 min at 121°, and use 
to prep, dilns of medicants. 

(e) Asparagine soln. — Make stock supply of 0.1% aspar- 
agine ("Bacto") soln in H 2 in erlenmeyer of convenient size, 
plug with cotton, and sterilize 20 min at 121°. Use to cover 
metal carriers for sterilization and storage. 

(f) Sodium hydroxide soln. — Approx. \N (4%). (For clean- 
ing metal carriers before use.) 

6. Apparatus 

(a) Glassware. — As in 955.11B(a). Also: straight side Pyrex 
test tubes, 20 X 150 mm; 15 X 1 10 mm petri dishes; 100 mL, 
300 mL, and 1 L erlenmeyers. Sterilize petri dishes in closed 
metal containers. Use 25 x 150 mm straight side tubes for 
disinfectant soln. (Smaller tubes can give high percentage of 
false positives when sides are touched.) 

(b) Water bath and racks.— See 955.11B(b) and (c). 

(c) Transfer loops and needles. — (7) See 955.11B(d). (2) 
Make 3 mm right angle bend at end of 50-75 mm nichrome 
wire No. 18 B&S gage. Have other end in suitable holder (glass 
or Al rod). 

(d) Carriers. — Polished stainless steel cylinders (penicillin 
cups), 8 ± 1 mm od, 6 ± 1 mm id, length 10 ± 1 mm, of 
type 304 stainless steel, SS 18-8. (Obtainable from S. & L. 
Metal Products Corp., 58-29 57 Drive, Maspeth, NY 11378.) 



'The use-dilution methods for testing disinfectants, 955.14, 955A5, and 964.02, 

are technique-sensitive and may produce questionable results unless conducted 
by experienced, trained analysts under strictly controlled conditions. Users of 
the methods are advised to consult the following reports of recent studies for 
current scientific data and interpretation: JAOAC 69, 1003(1986); 70, 635(1987); 
70, 903(1987); 71, 9(1988); 71, 288(1988); 71, 868(1988); 71, 1187(1988). 
Infect. Control 8, 501(1987). 



136 



Disinfectants 



AOAC Official Methods of Analysis (1990) 



Discard cylinders that are visibly damaged (dull, chipped, 
dented, or gouged). Biologically screen remaining cylinders 
by performing use-dilution test with Staphylococcus aureus 
ATCC 6538 and 500 ppm alkyldimethylammonium chloride 
with alkyl chain distribution C14, 50%; C12, 40%; C16, 10% 
(e.g., BTC-835 Onyx Chemical Co., Jersey City, NJ 07302). 
Discard those cylinders giving pos. results in screening pro- 
cedure. In subsequent testing of samples, cylinders in tubes 
showing growth must be rescreened and may not be reused 
unless screen tests result in no growth. 

(e) Petri dishes. — Have available ca 6 sterile petri dishes 
matted with 2 layers of S&S No. 597 or Whatman No. 2, 9 
cm filter paper. 

(f) Pipets. — Use only disposable pipets. (Reusable pipets 
may have residues or chips.) 

C. Operating Technic 

Soak ring carriers overnight in IN NaOH, rinse with tap 
H 2 until rinse H 2 is neut. to phthln, then rinse twice with 
distd H 2 0; place cleaned ring carriers in multiples of 10 in 
cotton-plugged erlenmeyers or 25 x 150 mm cotton plugged 
Pyrex test tubes, cover with asparagine soln, 955.14A(e), ster- 
ilize 20 min at 121°, cool, and hold at room temp. Vortex- 
mix nutrient broth test culture 3-4 s and let stand 10 min at 
room temp, before continuing. Transfer 20 sterile ring car- 
riers, using flamed nichrome wire hook, into 20 mL 38-54 hr 
nutrient broth test culture in sterile 25 X 150 mm medication 
tube. One or 2 addnl carriers may be added at same inoculum 
rate to serve as reserves. Carriers that fall over in petri dishes 
cannot be used in test. After 15 min contact period remove 
cylinders, using flamed nichrome wire hook, shake carrier vig- 
orously against side of tube to remove excess culture, and place 
on end in vertical position in sterile petri dish matted with filter 
paper, 955.14B(e), making sure that carriers do not touch to 
prevent improper drying. Cover and place in incubator at 37° 
and let dry 40 min. Hold broth culture for detn of its resistance 
to phenol by phenol coefficient method, 955. 11C. 

From 5% stock phenol soln (1-20) make 1-90 and 1-100 
dilns directly into medication tubes. Place tube for each diln 
in H 2 bath and let come to 20° (10 min). Make stock soln 
of germicide to be tested in sterile g-s cylinder. From this soln 
make 10 mL dilns to be tested, depending upon phenol coef- 
ficient found and /or claimed against S. typhi at 20°, directly 
into each of ten 25 X 150 mm medication tubes; place the 10 
tubes in H 2 bath at 20° and let come to temp. Prep, diln of 
germicide to be tested by diln in sterile H 2 0, 955.14A(d). Diln 
of sample should be made using >1.0 mL of sample. Use v/ 
v dilns for liq. products and w/v dilns for solids. Round to 2 
decimal places toward a stronger product. To ensure stable 
product, soln should be prepd <3 hr prior to use. Place tubes 
in 20° H 2 bath >10 min. Det. diln to be tested by multiplying 
phenol coefficient number found and/or claimed by 20 to det. 
number of parts H 2 in which 1 part germicide is to be in- 
corporated. This detn is not required when disinfectant under 
test yields phenol coefficient that cannot be converted validly 
to presumptive use-diln, or when analyst dets that use-diln range 
can be found without resort to phenol coefficient test. 

Add 0.5 mL of test culture suspension to 1-90 diln of phenol 
control; after 30 sec interval, add 0.5 mL to 1-100 diln of 
control, using sterile cotton-plugged pipets. After adding cul- 
ture, agitate tubes gently but thoroly to distribute bacteria evenly, 
and replace in bath; 5 min after seeding first medication tube, 
transfer 1 loopful of mixt. of culture and dild phenol from 
medication tube to corresponding subculture tube. After 30 sec, 
transfer loopful from second medication tube; 5 min after mak- 
ing first set of transfers begin second set of transfers for 10 
min period; and finally repeat for 15 min period. Use technic 
of loop sampling, flaming loop and mouths of tubes, and ag- 



itating medication and subculture tubes as in phenol coefficient 
method, 955. 11C. Incubate subcultures 48 hr at 37° and read 
results. Resistance in 48-54 hr culture of S. choleraesuis should 
fall within range specified for 24 hr culture of S. typhi in phenol 
coefficient method. 

Without touching sides of tube with contaminated carrier or 
hook, either when placing carrier in tube or when withdrawing 
hook, add 1 contaminated dried cylinder carrier at 1 min in- 
tervals to each of the .10 tubes of use-diln of germicide to be 
tested. (Note: Proper execution of transfer step is one of the 
most critical, technique-sensitive areas of method. False pos- 
itives will result if sides of tube are touched.) Thus, by time 
10 tubes have been seeded, 9 min will have elapsed, plus 1 
min interval before transfer of first carrier in series to individ- 
ual tube of subculture broth. This interval is const for each 
tube with prescribed exposure period of 10 min. The 1 min 
interval between transfers allows adequate time for flaming and 
cooling nichrome wire hook and making transfer in manner so 
as to drain all excess medication from carrier by shaking car- 
rier against side of tube. Shorter intervals may be used in add- 
ing and removing carriers if 2 alternately flamed and cooled 
hooks are used. Individual manipulation of carriers is required; 
use of semiautomated ring carrier is prohibited. (Note: Above 
step is one of the most critical, technique- sensitive areas of 
method. False positives can result from transfer of live organ- 
isms to sides of tube due to aerosol formation.) Flame lips of 
medication and subculture tubes in conventional manner. Im- 
mediately after placing carrier in medication tube, swirl tube 
3 times before placing it back in bath. Thoroly shake subcul- 
ture tubes, incubate 48 hr at 37°, and report results as + (growth) 
or — (no growth) values. Growth in tubes should be checked 
by gram stain to ensure that no contamination is present. Check 
>:20% of pos. tubes. Confirm all pos. results by duplicate test- 
ing to assure against false pos. tests. 

Where there is reason to suspect that lack of growth at con- 
clusion of incubation period may be due to bacteriostatic ac- 
tion of medicant adsorbed on carrier that has not been neutzd 
by subculture medium used, transfer each ring to new tube of 
sterile medium and reincubate for addnl 48 hr at 37°. Where 
soln under test is such that material adsorbed on ring carriers 
and transferred into subculture medium makes it unsuitable for 
growth of test organism, as may be case with coned acids and 
alkalies, products carrying antibiotics, and wax emulsions, 
transfer each ring to new tube of sterile medium 30 min after 
initial transfer and incubate both primary and secondary sub- 
culture tubes 48 hr at 37°. Results showing no growth on all 
10 carriers will confirm phenol coefficient number found. Re- 
sults showing growth on any of the 10 carriers indicate phenol 
coefficient number to be unsafe guide to diln for use. In latter 
case, repeat test, using lower dilns (higher conens) of germi- 
cide under study. Max. diln of germicide which kills test or- 
ganism on 10 carriers in 10 min interval represents presumed 
max. safe use-diln for practical disinfection. 

Refs.: J. Bacteriol. 49, 526(1945). Am. J. Vet. Res. 9, 
104(1948). JAOAC 36, 466(1953); 70, 318(1987); 71, 
117(1988); 72, 116(1989). 



955.15 Testing Disinfectants against 

Staphylococcus aureus 

Use-Dilution Method 

First Action 1953 

Final Action 1959 

Repealed First Action 1988 

Proceed as in 955. 14C except change phenol dilns and test 
organism to those specified in 955.12. Use 48-54 hr culture 



AOAC Official Methods of Analysis (1990) 



Chlorine 



137 



of Staph, aureus FDA 209, ATCC No. 6538, having at least 
resistance specified for 24 hr culture at 20° in phenol coeffi- 
cient method, 955.12. Obtain organism annually, directly from 
ATCC. Prior to beginning use-dilution test, vortex-mix nu- 
trient broth culture as in 955.14. Results showing growth on 
any of 10 carriers indicate that diln is too high for use in dis- 
infecting where pyogenic bacteria must be killed. In such cases 
repeat test, using lower dilns (higher concns). Max. diln of 
germicide which kills both this test organism and S. choler- 
aesuis on 10 carriers in 10 min interval represents max. pre- 
sumed safe use-diln for disinfecting in hospitals, clinics, and 
other places where pyogenic bacteria have special signifi- 
cance. 

Note: While killing in 10 of 10 replicates specified provides 
reasonably reliable index in most cases, killing in 59 of 60 
replicates is necessary for confidence level of 95%. 

Refs.: J. Bacteriol. 49, 526(1945). Am. J. Vet. Res. 9, 
104(1948). JAOAC 36, 466(1953); 70, 318(1987); 71, 
117(1988); 72, 116(1989). 



964.02 Testing Disinfectants against 

Pseudomonas aeruginosa 

Use-Dilution Method 
First Action 1964 

Proceed as in 955. 14C. Use 48-54 hr nutrient broth culture 
Ps. aeruginosa PRD 10 (ATCC 15442). Carry stock culture 
on BBL CTA (cystine trypticase agar) in stab culture incubated 
48 hr at 37° and stored at 5° with transfer every 30 days. Trans- 
fer nutrient broth test cultures daily for 30-day intervals with 
incubation at 37°. Make fresh transfer from stock culture every 
30 days. Do not shake 48-54 hr test culture but decant liq. 
culture aseptically, leaving pellicle behind, to obtain 20 mL 
culture for inoculating 20 carriers in medication tube. 

Proceed with vortex-mixing as in 955. 14C prior to use of 
culture. Alternatively, pellicle may be carefully suctioned off, 
and culture can be poured into clean, sterile tube before vor- 
tex-mixing. Any disruption of pellicle resulting in dropping, 
breaking up, or stringing of pellicle in culture before or during 
its removal renders that culture unusable in use-dilution test. 
This is extremely critical because any pellicle fragments re- 
maining will result in uneven clumping and layering of or- 
ganism on cylinder, allowing unfair exposure to disinfectant 
and causing false pos. results. 

Refs.: J. Bacteriol. 49, 526(1945). Am. J. Vet. Res. 9, 
104(1948); JAOAC 47, 29, 176(1964); 70, 318(1987); 
72, 116(1989). 



OTHER TESTS 

955.16 Chiorine (Available) in Disinfectants 
Germicidal Equivalent Concentration 
Final Action 

(Applicable to H 2 0-miscible disinfectants for detg available CI 
germicidal equiv. concns with products offered for use as san- 
itizing rinses for previously cleaned nonporous surfaces, es- 
pecially where speed of action and capacity are essential con- 
siderations) 

A. Reagents 

Use reagents specified in 955. 11 A and 955.11B(e) and (f), 
and in addn: 

(a) Sterile distilled H 2 0.—See 955.14A(d). 

(b) Sterile phosphate buffer soln. — pH 8.0. Add 97.5 mL 



soln contg 11.61 g anhyd. K 2 HP0 4 in 1 L H 2 to 2.5 mL soln 
contg 9.08 g anhyd. KH 2 P0 4 in 1 L H 2 and autoclave 20 
min at 121° in cotton-plugged erlenmeyer. 

(c) NaOCl std stock soln. — Approx. 5%. Store NaOCl stock 
soln in tightly closed bottle in refrigerator, and det. exact 
available CI concn at frequent intervals by As 2 3 titrn: Trans- 
fer 20 mL sample to 1 L vol. flask and dil. to vol. Pipet 50 
mL aliquot of mixt. into 200 mL erlenmeyer. Add excess As 2 3 
soln and then decided excess NaHC0 3 . Titr. excess As 2 3 with 
std 1 soln, using starch soln (mix ca 2 g finely powd. potato 
starch with cold H 2 to thin paste; add ca 200 mL boiling 
H 2 0, stirring const., and immediately discontinue heating; add 
ca 1 mL Hg, shake, and let soln stand over Hg), or use the 1 
as its own indicator. Subtract vol. 1 soln, corrected to 0.L/V, 
from vol. As 2 3 soln used, and from this value and sp gr of 
soln, calc.% NaOCl. 

1 mL O.liV As 2 3 = 0.003722 g NaOCl 

(d) Test organisms.— Use S. typhi ATCC No. 6539 or Staph, 
aureus ATCC No. 6538 or both. 

B. Apparatus 
See 955. 11B. 

C. Operating Technic 

Det. resistance of test culture to phenol as in 955.11, and 
use cultures with resistance specified. Prep., in sterile g-s cyl- 
inders, NaOCl solns contg 200, 100, and 50 ppm available CI 
in sterile buffer soln, 955.16A(b). Transfer 10 mL of each soln 
to 25 x 150 mm medication tubes, place tubes in 20° H 2 
bath, and let come to temp. 

Starting with tube contg 200 ppm available CI, add 0.05 mL 
test culture prepd as in 955.11B(e), shake, and return to H 2 
bath. After 1 min, make transfer to tube of appropriate sub- 
culture medium, 955.11A(d), using flamed 4 mm loop. At 1.5 
min, add another 0.05 mL culture to the 200 ppm CI soln, 
shake, and return to bath. After addnl 1 min interval (2.5 min 
in test), make second subculture in same manner, and in 30 
sec, or at 3 min time in test, add another 0.05 mL culture, 
shaking and returning to H 2 bath. After another 1 min in- 
terval (4 min in test), make another transfer to tube of sub- 
culture medium. 

Repeat operation to give total of 10 added increments. This 
requires total time of 14.5 min for each soln and addn of 0.5 
mL total culture with subculture at std 1 min intervals after 
addn of culture aliquots. At conclusion of test shake all sub- 
culture tubes and incubate 48 hr at 37°. 

Repeat operation with solns contg 100 and 50 ppm available 
CI. Prep, soln of germicide to be tested at concn recommended 
or selected for study in sterile H 2 in g-s graduate. Transfer 
10 mL to 25 x 150 mm medication tubes, place in H 2 bath, 
and let come to temp. Repeat operation with this soln. 

To be considered equiv. in disinfecting activity to 200 ppm 
available CI, unknown germicide must show absence of growth 
in as many consecutive tubes of subculture tube series as 200 
ppm available CI std. Det. activity equiv. to 100 and 50 ppm 
available CI in same manner. See example, Table 955.16. 

In this example, 25 ppm soln of germicide X could be con- 
sidered equiv. to 200 ppm soln of available CI, and 20 ppm 
soln equiv. to 100 ppm of available CI, but 10 ppm soln of 
germicide X would not be considered equiv. in germicidal ac- 
tivity to 50 ppm of available CI. 

Draw conclusions relative to germicidal equiv. concns only 
when resistance of test culture to NaOCl control is such that 
^1 neg. increment is obtained at 50 ppm concn and 1 pos. 
increment is obtained at 200 ppm level. 

Refs.: Soap Sanit. Chem. 27, No. 2, 133(1951). JAOAC 38, 
274(1955); 40, 755(1957). 



138 



Disinfectants 



AOAC Official Methods of Analysis (1990) 



Table 955.16 Example for Determination of Chlorine Germicidal Equivalent Concentration 



No growth 















Subculture Series 












Concn, ppm 






















Germicide 


Avail. CI 


1 


2 


3 


4 


5 


6 


7 


8 


9 


10 




200 


- 


__ 


- 


- 


_ 


+ 


+ 


+ 


+ 


+ 


NaOCI control 


100 


~ 


- 


- 


+ 


+ 


+ 


+ 


+ 


+ 


+ 




50 


- 


~- 


+ 


+ 


+ 


+ 


+ 


+ 


+ 


+ 




25 


- 


_ 


- 


_ 


_ 


+ 


+ 


+ 


+ 


+ 


Unknown (X) 


20 


- 


- 


_ 


- 


+ 


+ 


+ 


+ 


+ 


+ 




10 


— 


+ 


+ 


+ 


+ 


+ 


+ 


+ 


+ 


+ 



growth 



955.17 Fungicidal Activity of Disinfectants 
Using Trichophyton mentagrophytes 
Final Action 

(Applicable for use with H 2 0-miscible type fungicides used to 
disinfect inanimate objects) 

A. Test Organism 

Use as test fungus typical strain of Trichophyton mentagro- 
phytes isolated from dermatophytosis of foot. Strain must spo- 
rulate freely on artificial media, presence of abundant conidia 
being manifested by powdery appearance on surface of 10-day 
culture, particularly at top of agar slant, and confirmed by mi- 
croscopic examination. Conidia-bearing mycelium should peel 
easily from surface of glucose agar. Conidia of required re- 
sistance survive 10 min exposure at 20° to phenol diln of 1 :70, 
but not to one of 1:60. Strain No. 640, ATCC No. 9533, is 
suitable. 

B. Culture Medium 

Carry fungus on agar slants of following composition: Glu- 
cose 2%, Neopeptone (Difco) 1%, agar 2%, adjusted to pH 
6.1-6.3. Use same culture medium to prep, cultures for ob- 
taining conidial suspension, and use fluid medium of same nu- 
trient composition (without agar) to test survival and viability 
of conidia after exposure to fungicide. 

C. Care of Fungus Strain 

Store stock culture of fungus on glucose agar slants at 2— 
5°. At intervals <3 months, transfer to fresh agar slants, in- 
cubate 10 days at 25-30°, and store at 2-5° until next transfer 
period. Do not use culture that has been kept at or above room 
temp. >10 days as source of inoculum for culture. (Cultures 
may be kept at room temp, to preserve strain and to inoculate 
cultures if transferred at intervals <10 days.) 

D. Preparation of Conidial Suspension 

Prep, petri dish cultures by planting inoculum at center of 
agar plate and incubating culture at 25-30° for >10, but <15 
days. Remove mycelial mats from surface of 5 agar plate cul- 
tures, using sterile spatula or heavy flattened wire. Transfer to 
heat-sterilized glass tissue grinder, 966.04B(e), and macerate 
with 25 mL sterile physiological NaCl soln (0.85% NaCl), or 
to heat- sterilized erlenmeyer contg 25 mL sterile saline with 
glass beads, and shake thoroly. Filter suspension thru sterile 
absorbent cotton to remove hyphal elements. Est. density of 
conidial suspension by counting in hemacytometer and store 
at 2-10° as stock spore suspension (125-155 X 10 6 conidia/ 
mL) for <4 weeks for use in prepg test suspensions of conidia. 
Stdze test conidial suspensions as needed by dilg stock spore 
suspension with physiological NaCl soln so that it contains 5 
x 10 6 conidia/mL. 

E. Operating Technic 

Prep, dilns of fungicide. (Tests are similar to those de- 
scribed in 955. 11C.) Place 5 mL of each fungicide soln and 



of phenol control solns in 25 X 150 mm test-culture tubes, 
arrange in order of ascending dilns, place tubes in 20° H 2 
bath, and let come to temp. With graduated pipet, place 0.5 
mL spore suspension in first tube of fungicidal soln, shake, 
and immediately replace in H 2 bath; 30 sec later add 0.5 mL 
conidial suspension to second tube. Repeat at 30 sec intervals 
for each fungicidal diln. If more convenient, run test at 20 sec 
intervals. After 5, 10, and 15 min exposure to fungicide, re- 
move sample from each conidia-fungicide mixt. with 4 mm 
loop and place in 10 mL glucose broth, 955. 17B. To eliminate 
risk of faulty results due to possible fungistatic action, make 
subtransfers from the initial glucose broth subculture tubes to 
fresh tubes of glucose broth, using the 4 mm loop before in- 
cubation, or make initial subcultures in glucose broth contg 
either 0.05% Na thioglycolate, 1.5% isooctylphenoxy-poly- 
ethoxy-ethanol, or mixt. of 0.07% lecithin (Asolectin, Asso- 
ciated Concentrates, Inc., 32-34 61st St, Woodside, NY 11377) 
and 0.5% polysorbate 80 (Tween 80), whichever gives lowest 
result. Incubate inoculated tubes at 25-30°. Read final results 
after 10 days, altho indicative reading can be made in 4 days. 

Note: Highest diln that kills spores within 10 min is com- 
monly considered as highest diln that could be expected to 
disinfect inanimate surfaces contaminated with pathogenic fungi. 

Refs.: Arch. Dermatol. Syphilol. 28, 15(1933). J. Bacterid. 
42, 225(1941); 47, 102(1944). JAOAC 37, 616(1954); 
38, 274(1955); 56, 308(1973). 



960.09 Germicidal and Detergent 

Sanitizing Action of Disinfectants 
Final Action 

(Suitable for detg min. concn of chem. that can be permitted 
for use in sanitizing prec leaned, nonporous food contact sur- 
faces. Min. recommended starting concn is 2-4 x this concn. 
Test also dets max. water hardness for claimed conens. As 
control, check accuracy of hard-water tolerance results with 
pure C14 alkyl dimethyl benzyl NH 4 chloride at 700 and 900 
ppm hardness, and pure C 16 alkyl dimethyl benzyl NH 4 chlo- 
ride (Cetalkonium Chloride, at 400 and 550 ppm hardness, 
expressed as CaC0 3 .) 

A. Reagents 

(a) Culture media. — (/) Nutrient agar A. — Boil 3 g beef 
ext, 5 g peptone (Bacto, from Difco or equiv.; special grades 
must not be used), and 15 g salt- free agar in 1 L H 2 0. Do not 
use premixed, dehydrated media. Tube, and autoclave 20 min 
at 121°. Use for daily transfer of test culture. (2) Nutrient agar 
B. — Prep, as above but use 30 g agar. Use for growing test 
cultures in French square bottles. (3) Nutrient agar {AOAC), — 
See 955.11A(c). Use for prepg stock culture slants. 

(b) Subculture media. — (J) Use tryptone glucose ext agar 
(Difco), adding 25 mL stock neutralize^ (c)/L. (2) Tryptone 
glucose ext agar (Difco). 



AOAC Official Methods of Analysis (1990) 



Sanitizing Action 



139 



Table 960.09A Percent Light Transmission at Various Wavelengths Corresponding to Bacterial Concentrations 







% 


Light Transmission with Filters, 


nm 






Av. Bacteria] 


370 


420 


490 


530 


550 


580 


650 


Count/mL 


7.0 


4.0 


6.0 


6.0 


6.0 


7.0 


8.0 


13.0 x 10 9 


8.0 


5.0 


7.0 


7.0 


7.0 


8.0 


9.0 


11.5 


9.0 


6.0 


8.0 


8.0 


8.0 


9.0 


10.0 


10.2 


10.0 


7.0 


9.0 


9.0 


9.0 


11.0 


11.0 


8.6 


11.0 


8.0 


10.0 


10.0 


10.0 


12.0 


13.0 


7.7 


13.0 


9.0 


12.0 


12.0 


12.0 


13.0 


15.0 


6.7 



(c) Neutralizer stock soln. —Mix 40 g Asolectin (Associ- 
ated Concentrates, 32-34 61st St, Woodside, NY 11377), 280 
mL polysorbate 80, and 1.25 mL phosphate buffer, (e); dil. 
with H 2 to 1 L and adjust to pH 7.2. Dispense in 100 mL 
portions and autoclave 20 min at 121°. 

(d) Neutralizer blanks. — For use with <200 ppm quater- 
nary NH 4 compd. Mix 100 mL neutralizer stock soln, (c), 25 
mL 0.25M phosphate buffer stock soln, (e), and 1675 mL H 2 CX 
Dispense 9 mL portions into 20 x 150 mm tubes. Autoclave 
20 min at 121°. 

(e) Phosphate buffer stock soln. — 0.25M. Dissolve 34.0 g 
KH 2 P0 4 in 500 m'L H 2 0, adjust to pH 7.2 with IN NaOH, 
and dil. to 1 L. 

(f) Phosphate buffer dilution water. — Add 1 .25 ml 0.25M 
phosphate buffer stock soln, (e), to 1 L H 2 and dispense in 
99 mL portions. Autoclave 20 min at 121°. 

(g) Test organisms. — Use Escherichia coll ATCC No. 1 1229 
or Staphylococcus aureus ATCC 6538. Incubate 24 and 48 hr, 
resp. Maintain stock cultures on nutrient agar (AOAC), (a)(5), 
at refrigerator temp. 

B. Resistance to Phenol of Test Cultures 

Det. resistance to phenol at least every 3 months by 955.11. 
Resistance of E. coli should be equiv. to that specified for S. 
typhi in 955.1 ID and that for Staph, aureus equiv. to that 
specified for this organism in 955.12. 

C. Apparatus 

(a) Glassware. — 250 mL wide-mouth erlenmeyers; 100 mL 
graduate; Mohr, serological, and/or bacteriological (APHA 
specification) pipets; 20 x 150 mm test tubes. Sterilize at 180° 
in hot air oven ^2 hr. 

(b) Petri dishes. — Sterile. 

(c) French square bottles. — 175 mL, borosilicate (Thomas 
Scientific). Use of other containers will give variable results. 

(d) Water bath.— Controlled at 25°. 

D. Preparation of Culture Suspension 

From stock culture inoculate tube of nutrient agar A, 
960.09A(a)(/), and make >3 consecutive daily transfers (<30), 
incubating transfers 20-24 hr at 35-37°. Do not use transfers 
>30 days. If only 1 daily transfer has been missed, no special 
procedures are required; if 2 daily transfers are missed, repeat 
with 3 daily transfers. 

Prep. 175 mL French square culture bottles contg 20 mL 
nutrient agarB, 960.09 A(a)(2), autoclave 20 min at 121°, and 
let solidify with bottle in horizontal position. Inoculate culture 
bottles by washing growth from slant with 5 mL phosphate 
buffer diln H 2 0, 960.09A(f ), into 99 mL phosphate buffer diln 
H 2 0, and adding 2 mL of this suspension to each culture bot- 
tle, tilting back and forth to distribute suspension; then drain 
excess liq. Incubate 18-24 hr at 35-37°, agar side down. Re- 
move culture from agar surface of 4 or more bottles, using 3 
mL phosphate buffer diln H 2 and glass beads in each bottle 
to suspend growth. Filter suspension thru Whatman No. 2 pa- 
per pre wet with 1 mL sterile phosphate buffer, and collect in 
sterile tube. (To hasten filtration, rub paper gently with sterile 
policeman.) Stdze suspension to give av. of 10 x 10 9 organ- 



isms/mL by diln with sterile phosphate buffer diln H 2 0, 
960.09A(f). 

If Lumetron colorimeter is used, dil. suspension in sterile 
Lumetron tube to give % T according to Table 960. 09 A. 

If McFarland nephelometer and BaS0 4 stds are used, select 
7 tubes of same id as that contg test culture suspension. Place 
10 mL of each suspension of BaS0 4 , prepd as indicated in 
Table 960.09B, in each tube and seal tube. Stdze suspension 
to correspond to No. 4 std. 

E. Synthetic Hard Water 

Prep. Soln J by dissolving 31.74 g MgCl 2 (or equiv. of hy- 
drates) and 73.99 g CaCl 2 in boiled distd H 2 and dilg to 1 
L. Prep. Soln 2 by dissolving 56.03 g NaHC0 3 in boiled distd 
H 2 and dilg to 1 L. Soln 1 may be heat sterilized; Soln 2 
must be sterilized by filtration. Place required amt Soln 1 in 
sterile 1 L flask and add >600 mL sterile distd H 2 0; then add 
4 mL Soln 2 and dil. to 1 L with sterile distd H 2 0. Each mL 
Soln 1 will give a water equiv. to ca 100 ppm of hardness 
calcd as CaC0 3 by formula: 

Total hardness as ppm CaC0 3 

= 2.495 x ppm Ca + 4.115 x ppm Mg 

pH of all test waters <2000 ppm hardness should be 7.6- 
8.0. Check prepd synthetic waters chemically for hardness at 
time of tests, using following method or other methods de- 
scribed in 14th ed. of Standard Methods for the Examination 
of Water, Sewage, and Industrial Wastes. 

F. Hardness Method 

(a) EDTA std soln.— Dissolve 4.0 g Na 2 H 2 EDTA.2H 2 and 
0.10 g MgCl 2 .6H 2 in 800 mL H 2 and adjust by subsequent 
diln so that 1 mL of soln is equiv. to 1 mg CaC0 3 when titrd 
as in (c). Check EDTA soln after prepn or, if com. purchased, 
against CaC0 3 std at least every 2 months. 

(b) Calcium std soln. — 1 mL = 1 mg CaC0 3 . Weigh 1.00 
g CaC0 3 , dried overnight or longer at 105°, into 500 mL er- 
lenmeyer and add dil. HC1 thru funnel until CaCO^ is dis- 
solved. Add 200 mL H 2 0, boil to expel C0 2 , and cool. Add 
few drops Me red indicator and adjust color to intermediate 
orange with dil. NH 4 0H or HC1 as required. Transfer quant, 
to 1 L vol. flask and dil. to vol. 

(c) Determination. — Dil. 5-25 mL sample (depending on 
hardness) to 50 mL with H 2 in erlenmeyer or casserole. Add 
1 mL buffer soln (67.5 g NH 4 C1 and 570 mL NH 4 OH dild to 



Table 960. 09B Preparation of BaS0 4 Suspensions Corre- 
sponding to Bacterial Concentrations 





2% BaCI 2 


1% H 2 S0 4 


Av. Bacterial 


Std No. 


Soln, mL 


(v/v) Soln, mL 


Count/mL 


1 


4.0 


96.0 


5.0 x 10 9 


2 


5.0 


95.0 


7.5 


3 


6.0 


94.0 


8.5 


4 


7.0 


93.0 


10.0 


5 


8.0 


92.0 


12.0 


6 


10.0 


90.0 


13.5 


7 


12.0 


88.0 


15.0 



140 



Disinfectants 



AOAC Official Methods of Analysis (1990) 



1 L with H 2 0), 1 mL inhibitor (5.0 g Na 2 S.9H 2 or 3.7 g 
Na 2 S.5H 2 dissolved in 100 mL H 2 0), and 1 or 2 drops in- 
dicator soln (0.5 g Chrome Black T in 100 mL 60-80% al- 
cohol). Titr. with EDTA std soln slowly, stirring continuously, 
until last reddish tinge disappears from soln, adding last few 
drops at 3-5 sec intervals. 

Hardness as mg CaC0 3 /L 

= (mL std soln x 1000)/mL sample 

G. Preparation of Samples 

Use composition declared or detd as guide to sample wt re- 
quired for vol. sterile H 2 used to prep. 20,000 ppm soln. 
From this stock diln, transfer 1 mL into 99 mL of the water 
to be used in test to give concn of 200 ppm. In making trans- 
fer, fill 1 mL pipet and drain back into stock soln; then refill, 
to correct for adsorption on glass. After mixing, discard 1 mL 
to provide 99 mL of the test water in 960.09H. 

H. Operating Technic 

Measure 99 mL water to be used in test ? contg bactericide 
at concn to be tested, into chem. clean, sterile, 250 mL wide- 
mouth erlenmeyer and place in const temp, bath until it reaches 
25°, or >20 min. Prep, duplicate flasks for each germicide to 
be tested. Also prep, similar flask contg 99 mL sterile phos- 
phate buffer diln H 2 0, 960.09A(f), as "initial numbers" con- 
trol. 

Add 1 mL culture suspension to each test flask as follows: 
Whirl flask, stopping just before suspension is added, creating 
enough residual motion of liq. to prevent pooling of suspen- 
sion at point of contact with test water. Add suspension mid- 
way between center and edge of surface with tip of pipet slightly 
immersed in test soln. Avoid touching pipet to neck or side of 
flask during addn. Transfer 1 mL portions of this exposed cul- 
ture to neutralizer blanks exactly 30 and 60 sec after addn of 
suspension. Mix well immediately after transfer. 

For "numbers control" transfer, add 1 mL culture suspen- 
sion to 99 mL sterile phosphate diln H 2 in same manner. In 
case of numbers control, plants need be made only immedi- 
ately after adding and mixing thoroly <30 sec. (It is advan- 
tageous to use milk pipets to add culture and withdraw sam- 
ples.) 

Plate from neutralizer tube to agar, using subculture medium 
960.09A(b)(7) for quaternary NH 4 compds and 960.09A(b)(2) 
with numbers control. Where 0.1 mL portions are planted, use 
1 mL pipet graduated in 0.1 mL intervals. For dilns to give 
countable plates, use phosphate buffer diln H 2 0, 960.09A(f). 
For numbers control, use following diln procedure: Transfer 1 
mL exposed culture (1 mL culture suspension transferred to 
99 mL phosphate buffer diln H 2 in H 2 bath) to 99 mL phos- 
phate buffer diln H 2 0, 960.09A(f), (diln J). Shake thoroly and 
transfer 1 mL diln 1 to 99 mL phosphate buffer diln H 2 0, 
960.09A(f), (diln 2). Shake thoroly and transfer 1 mL diln 2 
to 99 mL phosphate buffer diln H 2 (diln 3). Shake thoroly 
and transfer four 1 mL and four 0. 1 mL aliquots from diln 3 
to individual sterile petri dishes. 

For test samples, use following diln procedure: Transfer 1 
mL exposed culture into 9 mL neutralizer, 960.09A(d). Shake 
and transfer four 1 mL and four 0. 1 mL aliquots to individual 
sterile petri dishes. For numbers control, use subculture me- 
dium 960.09 A(b)(2); for tests with quaternary NH 4 compds, 
use medium 960.09A(b)(7). Cool agar to solidify, and then 
invert and incubate 48 hr at 35° before counting. 

/. Results 

To be considered valid, results must meet std effectiveness: 
99.999% reduction in count of number of organisms within 30 
sec. Report results according to actual count and % reduction 



over numbers control. Counts on numbers control for germi- 
cide test mixt. should fall between 75 and 125 X 10 6 /mL for 
% reductions to be considered valid. 

J. Sterility Controls 

(a) Neutralizer. — Plate 1 mL from previously unopened tube. 

(b) Water. — Plate 1 mL from each type of water used. 

(c) Sterile distilled water. —Plate 1 mL. 

After counting plates, confirm that surviving organisms are 
E. coli by transfer to brilliant green bile broth fermentation 
tubes or lactose broth and EMB agar; confirm Staph, aureus 
by microscopic examination. 

Refs.: Am. J. Public Health 38, 1405(1948). J. Milk Food 
Technol. 19, 183(1956). Fed. Regist. 21, 7020(1956). 
JAOAC 41, 541(1958); 56, 308(1973). 



961.02 Germicidal Spray Products 

as Disinfectants 

First Action 1961 
Final Action 1964 

(Suitable for detg effectiveness of sprays and pressurized spray 
products as spot disinfectants for contaminated surfaces) 

A. Reagents 

Use culture media and reagents specified in 955. 11 A, 
955.11B(e) and (f), and 955. 14A except that test organism 
Salmonella typhi is not used. 

Use as test organisms Trichophyton mentagrophytes ATCC 
No. 9533, prepd as in 955.17D, to which has been added 0.02 
mL octyl-phenoxy-polyethoxy-ethanol (Triton X 100, Rohm & 
Haas Co.)/ 10 mL suspension to facilitate spreading, Salmo- 
nella choleraesuis ATCC No. 10708, 955.14A(b), Staphylo- 
coccus aureus ATCC No. 6538, maintained as in 955.14A(b), 
and Pseudomonas aeruginosa. ATCC No. 15442, maintained 
as in 964.02. 

B. Apparatus 

Use app. specified in 955. 11B and 955. 14B, and in addn: 

(a) Capillary pipets. — 0.1 mL, graduated to deliver 0.01 
mL. Sterilize in air oven 2 hr at 180°. 

(b) Microscope slides. — Non-corrosive, 25 x 25 mm (1 x 
1"), or 18 X 36 mm glass slide. Sterilize by placing individual 
slides in petri dish matted with 2 pieces 9 cm filter paper 
(Whatman No. 2, or equiv.) in air oven 2 hr at 180°. 

(c) Bacteriological culture tubes. — Pyrex, 32 X 200 mm. 

(d) Metal forceps. — Sharp points, straight, 115 mm long. 

C. Operating Technic 

Thoroly shake 48 hr nutrient broth cultures of S. choler- 
aesuis and Staph, aureus and let settle 10 min. With sterile 
capillary pipet or sterile 4.0 mm loop, transfer 0.01 mL culture 
onto 1 sq in. sterile test slide in petri dish and immediately 
spread uniformly over entire area. Cover dish immediately and 
repeat operation until 12 slides have been prepd for each or- 
ganism. (Use 2 slides as control.) Dry all slides 30-40 min at 
37°. 

Spray 10 slides for specified time and distance. Hold each 
slide 10 min, drain off excess liq., and transfer slide to indi- 
vidual 32 X 200 mm tube contg 20 mL appropriate subculture 
medium, 955.11A(d), with flamed forceps. Shake culture tho- 
roly. If broth appears cloudy after 30 min, make subculture to 
fresh individual tubes of subculture broth. Transfer 2 un- 
sprayed slides, as viability controls, to individual subculture 
tubes in same manner. 

Incubate all tubes used for primary and secondary transfers 



AOAC Official Methods of Analysis (1990) 



Sporicidal Activity 



141 



48 hr at 37°. Read as + (growth) or - (no growth). Killing 
of test organisms in 10 of 10 trials is presumptive evidence of 
disinfecting action. 

Det. resistance of S. choleraesuis as in 955. 11C; with S. 
aureus as in 955.12; with Ps. aeruginosa as in 955.13; and 
with T. mentagrophytes as in 955. 17 A. 

If there is reason to believe that lack of growth in subtransfer 
tubes is due to bacteriostasis, inoculate all incubated subcul- 
ture tubes with loop needle inoculation of respective test cul- 
ture and reincubate. Growth of these inocula eliminates bac- 
teriostasis as cause of lack of growth. If there is question as 
to possibility of contamination as source of growth in subcul- 
ture tubes, make gram stains and/or subculture for identifi- 
cation, according to respective test culture. 

If fungicidal activity as well as germicidal activity is in- 
volved, use test suspension of 7\ mentagrophytes spores, 
955. 17D, and prep. 1.2 slides, using 0.01 mL std spore sus- 
pension, spraying and subculturing exactly as above. Make 
subcultures in glucose broth, 955. 17B, incubating 7 days at 
25-30°. 

Refs.: JAOAC 44, 422(1961); 50, 763(1967). Soap Chem. 
Spec. 38(2), 69(1962); 61, 400(1978). 



966.04 Sporicidal Activity of Disinfectants 

First Action 1966 
Final Action 1967 

(Suitable for detg sporicidal activity of liq. and gaseous chems. 
Applicable to germicides for detg presence or absence of spo- 
ricidal activity against specified spore-forming bacteria in var- 
ious situations and potential efficacy as sterilizing agent.) 

A. Reagents 

(a) Culture media. — (7) Soil extract nutrient broth. — Ext 
1 lb garden soil in 1 L H 2 0, filter several times thru S&S No. 
588 paper, and dil. to vol. (pH should be >5.2). Add 5 g beef 
ext. (Difco), 5 g NaCl, and 10 g peptone (Anatone, 
955.11 A(a)). Boil 20 min, dil. to vol., adjust with 17V NaOH 
to pH 6.9, and filter thru paper. Dispense in 10 mL portions 
into 25 x 150 mm tubes, and autoclave 20 min at 121°. Use 
this broth to propagate test culture of Bacilli. 

(2) Nutrient agar. — See 955.11A(c). Use slants of this me- 
dium to maintain stock culture of Bacilli. 

(3) Modified fluid thioglycolate medium USP. — Prep, as in 
955.11A(d)(2), except add 20 mL 17V NaOH to each L before 
dispensing for sterilization. Use this medium to subculture spores 
exposed to 2.5N HC1. For spores exposed to unknown ger- 
micides, use fluid thioglycolate medium, 955.11A(d)(2). 

(4) Soil extract-egg-meat medium.. — Add 1.5 g Bacto Egg- 
Meat Medium dehydrated (Difco No. 0042-17) to 25 X 150 
mm tube; then add 15 mL garden soil ext, (7), and sterilize 
20 min at 121°. Use this medium to propagate test cultures of 
Clostridia and maintain stock cultures of species of this genus. 

(b) Test organisms. — Use Bacillus subtilis, ATCC No. 
19659, or Clostridium sporo genes y ATCC No. 3584, for rou- 
tine evaluation. Method is also applicable for use with strains 
of B. anthracis, CI. tetani, or other spore forming species. 

(c) Dilute hydrochloric acid. — 2.57V. Use to det. resistance 
of dried spores. Stdze and adjust to 2.57V as in 936. 15B. 

B. Apparatus 

(a) Glassware. — Bacteriological culture tubes, unflared, 25 
x 150 mm; 100 mL g-s cylinders graduated in 1 mL divisions; 
65 mm id funnels; supply of 15 x 110 mm petri dishes matted 
with 2 sheets 9 cm S&S No. 597 or Whatman No. 2 filter 



paper. Sterilize all glassware and matted petri dishes 2 hr in 
air oven at 180°. 

(b) Water bath.— See 955.11B(b). 

(c) Racks.— See 955.116(c). 

(d) Transfer loop, hook, and forceps. — See 955.14B(c). 

(e) Tissue grinder. — Thomas Scientific, No. 3431-E20, Size 

B, or equiv. 

(f ) Suture loop carrier. — From spool of size 3 surgical silk 
suture (silk black braid A-59, USP, Ethicon, Inc., Rte 22, 
Sommerville, NJ 08876), prep, std loops by wrapping the silk 
around ordinary pencil 3 times, slipping coil so formed off end 
of pencil, and holding it firmly with thumb and index finger 
of left hand while passing another piece of suture through coil, 
knotting, and tying securely. Then shear off end of coil and 
knotted suture to within 2 mm. This should provide overall 
length of ca 65 mm of suture in 2-loop coil that can be con- 
veniently handled in ordinary aseptic transfer procedure. 

Ext loops in groups of 100-200 in Soxhlet extn app., using 
CHC1 3 , for 24 h. Air-dry 12-18 h at room temp, in hood. 
Place 100 loops in 100 mL 0.5N HC1 for 10 min or until all 
loops are completely submerged in soln. Decant, and rinse re- 
peatedly with distd H 2 for 15 min. Check rinse H 2 for ab- 
sence of HO, using litmus paper. Air-dry on filter paper mats 
under ambient conditions or in incubator. 

(g) Cylinder carriers . — " Penicy finders , M porcel ain , 8 ± 1 mm 
od, 6±1 mm id, 10± I mm long. (Available from Fisher Sci- 
entific Co., No. 7-907.) Sterilize 2 hr in 180° air oven. Wash 
used Penicylinders with Triton X-100 and rinse with H 2 4 
times. 

C. Operating Technic 

Grow all Bacilli in soil ext nutrient broth and all Clostridia 
in soil ext- meat-egg medium. Inoculate 3 tubes, using 1 loop 
stock culture, and incubate 72 hr at 37°. Place supply of suture 
loops and cylinder carriers in sep. petri dishes matted with 
filter paper, and sterilize 20 min at 121°. Use new loops for 
each test. Penicylinders must be free from chips or cracks. 
Filter CI. sporogenes thru funnel contg 2x5x5 cm sq piece 
of moist cotton or glass wool into sterile 25 x 150 mm test 
tubes, using same funnel. In prepg B. subtilis culture, pour 
tube of 72 hr culture into tissue grinder and macerate to break 
up pellicle. Filter thru sterile funnel contg moist cotton or glass 
wool into sterile 25 x 150 mm tube, repeating operation for 
other 2 tubes. Place 10 sterile suture loops or Penicylinders 
into each of 3 tubes contg 10 mL filtrate from 72 hr culture 
of CI. sporogenes, agitate, and let stand 10-15 min. Using 
this technic, contaminate 35 loops or cylinders. Place contam- 
inated suture loops and /or cylinders into petri dish matted with 
2 layers of filter paper. Drain. Proceed similarly for B. sub- 
tilis . 

Place the 35 suture loops or cylinders contaminated with CI. 
sporogenes or B. subtilis in vac. desiccator contg CaCl 2 and 
draw vac. of 69 cm (27") Hg for 20 min. Dry 24 hr under vac. 
(Spores dried and held under these conditions will retain re- 
sistance >1 days.) 

Transfer 10 mL 2.5AT HC1, 966.04A(c), into sterile 25 x 
150 mm tube. Place tube in 20° const temp. H 2 bath and let 
come to temp. Rapidly transfer 4 dried, contaminated loop or 
cylinder carriers to acid tube. Transfer remaining dried, con- 
taminated suture loop or cylinder carriers to tube of thiogly- 
colate subculture medium, 966.04A(a)(J), as viability control. 
After 2, 5, 10, and 20 min, withdraw individual loops or cyl- 
inders from acid and transfer to individual tubes of subculture 
medium. Rotate each tube vigorously 20 sec and resubtransfer. 
Incubate 21 days at 37°. Test spores should resist HC1 ^2 min, 
and many may resist HC1 for full 20 min. 

When testing sporicidal or sterilizing activity of gas, place 



142 



Disinfectants 



AOAC Official Methods of Analysis (1990) 



carriers in polyethylene bags or in petri dishes with lids ajar. 
Certain gases may require rehydration of spores before ex- 
posure to gas. Rehydrate spores on carriers by 1 hr immersion 
in H 2 0, using <20 mL H 2 0/6 carriers. Drain carriers 20 min 
on petri dishes matted with filter paper. After exposure to gas, 
remove carriers, using aseptic technic to subculture media as 
specified in next par. 

For aq. sporicides and sterilizers, place 10 mL product at 
diln recommended for use or under investigation into each of 
six 25 x 150 mm tubes. Place tubes in 20° H 2 bath and let 
come to temp. Using flamed forceps, place 5 suture loops or 
cylinders, contaminated with CI. sporogenes or 5. subtilis and 
dried 24 hr under vac, into each of the 6 tubes contg disin- 
fectant, using 2-min intervals for seeding each tube. Five su- 
ture loops or cylinders can be placed into each tube within 5 
sec. This seeding operation will take 10 min. After contact 
period specified for disinfectant has been achieved, remove 
suture loops or cylinders, using sterilized needle hook, from 
each tube of disinfectant to subculture medium or other sub- 
culture medium specified in 955. 11 A(d) (select medium contg 
most suitable neutralizer), placing 1 suture loop or cylinder per 
tube. Five cylinders can be removed within each 2 min inter- 
val. Flame transfer needle hook after each carrier has been 
transferred to subculture medium. After completing transfer, 
resubtransfer each suture loop or cylinder to fresh tube of 
thioglycolate medium and incubate 21 days at 37°. If no growth 
is observed after 21 days, heat-shock tubes 20 min at 80° and 
reincubate 72 hr at 37°. Report results as + (growth) or — (no 
growth) values. 

Killing in 59 of 60 replicates on 1 carrier at diln and time 
specified is considered evidence of sporicidal efficacy against 
1 test spore and for confidence level of 95%. Tests with both 
B. subtilis and CI. sporogenes, using 30 replicates with each 
of 2 carriers specified to provide min. of 120 carriers, are re- 
quired to presumptively support unqualified sporicidal claim 
or for presumptive evidence of sterilizing activity at concn, 
time, and conditions specified. For sporicidal claims, no more 
than 2 failures can be tolerated in this 120 carrier trial. For 
sterilizing claims, no failures can be tolerated. 

Refs.: JAOAC 36, 480(1953); 39, 480(1956); 40, 759(1957); 
49, 721(1966); 50, 194(1967); 56, 308(1973); 61, 

371(1978); 68, 279(1985). 



965.12 Tuberculocidal Activity 

of Disinfectants 

First Action 1965 

Final Action 1967 

Repealed First Action 1988 

(Suitable for detg max. tuberculocidal diln of disinfectants used 

on inanimate surfaces. This method has not been validated for 

glutaraldehyde-based products) 



/. Presumptive In Vitro Screening Test Using 
Mycobacterium smegmatis 

A. Reagents 

(a) Test organism. — Mycobacterium smegmatis (PRD No. 
1) (available from Microbiology Lab., U.S. Environmental 
Protection Agency, Benefits & Use Div., Bldg 306, BARC- 
East, Beltsville, MD 20705). Maintain on nutrient agar slants 
by monthly transfers. Incubate new stock transfer 2 days at 
37°; then store at 2-5°. From stock culture inoculate tubes of 
Proskauer-Beck broth, (b)(7), incubate 48 hr in slanting po- 
sition, carry 30 days, using 48 hr transfers, and use these 48 
hr cultures to start test cultures. Inoculate 1 or 2 tubes of Pros- 



kauer-Beck broth. Incubate 6-7 days at 37°. Incubate tubes 48 
hr in slanting position to provide max. surface aeration and 
then in upright position 4-5 days. Add 1.5 mL sterile 2.0% 
Bacto-Gelatin soln and homogenize culture with sterilized glass 
tissue grinder, 966.046(e). Adjust to 20% T at 650 nm with 
sterile Proskauer-Beck broth for use in testing. 

(b) Culture media. — (7) Modified Proskauer-Beck broth. — 
Dissolve 2.5 g KH 2 P0 4 , 5.0 g asparagine, 0.6 g MgS0 4 .7H 2 0, 
2.5 g Mg citrate, 20.0 mL glycerol, 0.0046 g FeCl 3 , and 0.001 
g ZnS0 4 .7H 2 in 1 L H 2 0. Adjust to pH 7.2-7.4 with IN 
NaOH. Filter thru paper, place 10 mL portions in sep. 20 x 
150 mm tubes, and sterilize 20 min at 121°, Use for propa- 
gating 48 hr test starter cultures and 6-7 day test cultures. 

(2) Subculture media. — Use (7) with addn of suitable neutzg 
agents such as purified lecithin (Azolectin) or Na thioglyco- 
late, where necessary. 

(3) Nutrient agar. — Prep, as in 955.11A(c). Use to main- 
tain stock culture. 

(4) Sterile distilled water.— See 955.14A(d). 

B. Apparatus 

(a) Glassware, water bath, transfer loops and needles, and 
petri dishes. —See 955.14B(a), (b), (c), and (e). 

(b) Carriers. —See 966.04B(g). 

C. Operating Technic 

Transfer 20 sterile Penicylinder carriers, using flamed ni- 
chrome wire hook, into 20 mL 6-7 day homogenized stdzd 
broth culture, 965.12A(a), in sterile 25 x 150 mm medicant 
tube. After 15 min contact, remove cylinders and place on end 
in vertical position in sterile petri dish matted with filter paper, 
955.14B(e). Cover and place in incubator at 37° and let dry 
>20 min but <60 min. This will provide dried test carriers in 
groups of 20 in individual petri dishes. With each group of 20 
carriers, add 1 dried cylinder at 30 sec intervals to each of 20 
tubes contg 10 mL diln of germicide to be tested (at 20° in 
FLO bath). Flame lips of medicant and subculture tubes. Im- 
mediately after placing carrier in medicant tube, swirl tube 3 
times before placing it back in H 2 bath. (Thus, by time 20 
tubes have been seeded, 9 min and 30 sec have elapsed, leav- 
ing 30 sec interval prior to subculturing series at 10 min ex- 
posure for each carrier. The 30 sec interval between transfers 
allows adequate time for flaming and cooling transfer hook 
and making transfer in manner so as to drain all excess med- 
icant from carrier.) Transfer carrier to 10 mL subculture me- 
dia, 965.12A(b)(2). Shake all subculture tubes thoroly and in- 
cubate 12 days at 37°. Report results as + (growth) or — (no 
growth). Where there is reason to suspect that results may be 
affected by bacteriostatic action of medicant carried over in 
subculture tubes, use suitable neutralizer in subculture media. 

Make >30 carrier exposures at each of 3 relatively widely 
spaced dilns of germicide under test between no response and 
total response diln levels. Calc. % of carriers on which or- 
ganism is killed at each diln. Using log % probit paper (3 cycle 
logarithmic normal No. Y3 213 HG, Codex Book Co., Inc., 
74 Broadway, Norwood, MA 02062), locate % kill points on 
diln lines employed (log scale). Draw best fitting straight line 
thru these 3 points and extend to intercept 99% kill line. Read 
diln line (log scale) at point of intercept. This is presumed 95% 
confidence end point for product. (Do not use presumptive test 
organism for checking validity of this presumptive end point.) 



//. Confirmative In Vitro Test for Determining 
Tuberculocidal Activity — First Action 1965 

D. Reagents 

(a) Culture media. — (7) Modified Proskauer-Beck me- 
dium.. — Prep, as in 966. 12 A (b)(7), and in addn, place 20 mL 



AOAC Official Methods of Analysis (1990) 



TUBERCULOCIDAL ACTIVITY 



143 



portions in 25 x 150 mm tubes. Use 10 mL portions for daily 
transfers of test cultures and 20 mL portions for subculturing 
porcelain cylinders. 

(2) Middlebrook 7H9 Broth Difco A.— Dissolve 4.7 g in 
900 mL H 2 contg 2 mL glycerol and 15.0 g agar. Heat to 
bp to dissolve completely. Distribute in 180 mL portions and 
autoclave 15 min at 121°, To each 180 mL sterile medium at 
45°, add 20 mL Middlebrook ADC Enrichment (Difco) under 
aseptic conditions and distribute in 10 mL portions in sterile 
20 X 150 mm tubes. Slant. Use to maintain test culture. 

(3) Middlebrook 7H9 Broth Difco B.— Dissolve 4.7 g in 
900 mL H 2 contg 2 mL glycerol and 1.0 g agar. Heat to bp 
to dissolve completely. Distribute in 18 mL portions in 25 x 
150 mm tubes, and autoclave 15 min at 121°. To each 18 mL 
sterile medium at 45° add 2 mL Middlebrook ADC Enrichment 
under aseptic conditions. Use to subculture for survival. 

(4) Kirchners Medium Difco. — Dissolve 13.1 g in 1 L H 2 
contg 20 mL glycerol and heat to bp to dissolve completely. 
Distribute in 18 mL portions in 25 x 150 mm tubes and au- 
toclave 15 min at 121°. If commercial medium is not available, 
add 5 g asparagine (Difco), 2.5 g Na citrate, 0.6 g MgS0 4 , 
2.5 g monopotassium phosphate, 1.5 g dipotassium phosphate, 
1 g Bacto agar in 1 L H 2 0, and add 20 mL glycerol. To each 
1 8 mL sterile medium at 50-55° add 2 mL Middlebrook ADC 
Enrichment under aseptic conditions. Use to subculture for 
survival. 

(5) TB Broth Base Difco {without Polysorbate 80). — Dis- 
solve 11.6 g in I L H 2 contg 50 mL glycerol and 1.0 g agar. 
Heat to bp to dissolve completely. Distribute in 18 mL por- 
tions in 25 X 150 mm tubes, and autoclave 15 min at 121°. 
To each 18 mL sterile medium at 50° add 2 mL Dubos Me- 
dium Serum (Difco) under aseptic conditions. Use to subcul- 
ture for survival. 

(b) Test organism. — Mycobacterium bovis (BCG) (Bionet- 
ics Research, Inc., 115 S Sangamon St, Chicago, IL 60607). 
Maintain stock cultures on culture medium (a)(2) by monthly 
or 6 weeks transfer. Incubate new stock transfer 15-20 days 
at 37° until sufficient growth is indicated; then store at 2-5°. 
From stock culture, inoculate tube of culture medium (a)(7) 
and incubate 21-25 days at 37°. Allow to remain quiescent 
until 21 -25th day. Make daily transfers from 21 day cultures. 
Transfer culture to heat-sterilized glass tissue grinder, add 1.0 
mL 0.1% Tween 80 in saline soln (Difco), grind, and dil. with 
culture medium (a)(7) to give 20% T at 650 nm. Use to in- 
oculate porcelain cylinders used in test. Tests will be satisfac- 
tory only when organism is killed on all 10 carriers by aq. 
phenol (1 +50) and shows survival after exposure to aq. phenol 
(1 + 75) control. Prep, dilns from 5% std phenol soln, 
955.116(f). 

(c) Sterile distilled water. — See 955.14A(d). 

(d) Normal horse serum. — Difco Laboratories. 

E. Apparatus 

(a) Glassware, water bath, transfer loops and needles, and 
petri dishes.— See 955.14B(a), (b), (c), and (e). 

(b) Carriers.— See 966.04B(g) o 

F. Operating Technic 

Soak ring carriers overnight in \N NaOH; rinse with tap 
H 2 and then with distd H 2 until distd H 2 is neut. to phthln; 
then rinse twice with distd H 2 0. Place clean ring carriers in 
multiple of 10 or 20 in capped erlenmeyer or 20 x 150 mm 
tubes. Autoclave 20 min at 121°, cool, and hold at room temp. 
Transfer 10 sterile ring carriers, using flamed wire hook, into 
enough (ca 15-20 mL) 21-25 day stdzd test culture, 
965.12D(b), in 25 X 150 mm medication tube. After 15 min 
contact period, remove cylinders, using flamed wire hook, and 
place on end in vertical position in sterile petri dish matted 



with filter paper, 955.14B(e). Cover, place in incubator at 37°, 
and let dry 30 min. 

Let 10 tubes contg 10 mL use-diln germicide sample to be 
tested come to 20° (or desired temp., if germicide use is rec- 
ommended for temp, other than room temp.) in H 2 bath and 
add 1 contaminated cylinder carrier at either 30 sec or 1 min 
intervals to each tube. (Thus, by time 10 tubes have been seeded, 

9 min will have elapsed, plus 1 min interval before transfer of 
first carrier in series to individual tube of 10 mL neutralizer 
appropriate for germicide tested, or 10 mL neutralizer blank, 
960.09A(d), if 1 min intervals are used. This interval is con- 
stant for each tube with prescribed exposure period of 10 min. 
Interval between transfers allows adequate time for flaming 
and cooling wire hook and making transfer in manner so as to 
drain all excess medication from carrier.) Transfer carrier to 

10 mL neutralizer appropriate for germicide tested, after ex- 
actly 10 min contact. Shake tube contg carrier in neutralizer 
thoroly and place carrier in tube contg 20 mL broth, 
965.12D(a)(7). From same tube, take 2 mL portions serum 
and place in any 2 of the subculture media, 965.12D(a)(J), 
(4), (5). Repeat this with each of the 10 carriers. Incubate 1 
tube of each subculture medium with 2 mL sterile serum as 
control. Where there is reason to suspect that germicide to be 
tested may possess bacteriostatic action, use suitable neutral- 
izer in lieu of serum. Shake each subculture tube thoroly, in- 
cubate 60 days at 37°, and report results as + (growth) or - 
(no growth). If no growth or only occasional growth is ob- 
served in subculture, incubate addnl 30 days before making 
final reading. Max. diln of germicide which kills test organism 
on the 10 carriers, and no growth in each of the 2 mL aliquots 
for 2 extra media, represents max. safe use-diln for practical 
tuberculocidal disinfection. 

Refs.: JAOAC 48, 635(1965); 50, 767(1967); 53, 860(1970); 
70, 318(1987). 



972.04 Bacteriostatic Activity of Laundry 
Additive Disinfectants 
First Action 1972 

(Applicable to antimicrobial products, recommended for use 
during laundering operations, which are intended to provide 
residual bacteriostatic treatment to laundered fabric. Method 
includes treatment of fabric with product and subsequent bac- 
teriostatic testing of treated fabric.) 

A. Reagents 

(a) Culture media. — (/) Nutrient broth. — See 955.11A(a). 

(2) Nutrient agar A. — See 955.11A(c). Use for monthly 
transfer of stock cultures. 

(3) Nutrient agar B. — Boil 3 g beef ext, 5 g peptone (An- 
atone), 8 g NaCl, and 10 g agar (Difco) in 1 L H 2 6. Transfer 
100 mL portions to erlenmeyers, and autoclave 20 min at 121°. 
Use for agar plate tests to evaluate bacteriostatic activity of 
treated fabric. See also (c). 

(b) Test organisms. — Use Staphylococcus aureus ATCC No. 
6538 and Klebsiella pneumoniae, aberrant ATCC No. 4352 
(formerly Escherichia coli), and maintain as in 955.11B(e). 

(c) 2,3,5-Triphenyl tetrazolium chloride. — Use as optional 
biological indicator. With S. aureus, use 0.15% soln; with K. 
pneumoniae, aberrant, use 0.25% soln. Autoclave each 20 min 
at 121°. Apply as in 972.04D. 

(d) Alkaline nonionic wetting agent. — Prep. aq. soln contg 
0.5% alkyl phenol poly glycol ether wetting agent and 0.5%' 
Na 2 C0 3 . Use to scour test fabric. 



144 



Disinfectants 



AOAC Official Methods of Analysis (1990) 



B. Apparatus 

(a) Test fabric. — 80 x 80 thread s/sq in. plain weave cotton 
print cloth, completely desized, bleached, and without bluing 
or optical brighteners. Scour before use by boiling ca 300 g 1 
hr in 3 L H 2 contg 1.5 g nonionic wetting agent and 1.5 g 
Na 2 C0 3 . Then rinse fabric, first in boiling H 2 and then in 
cold H 2 0, until all visual traces of wetting agent are removed. 
Air-dry and cut into long strip 5 cm (2") wide and weighing 
exactly 15 g. 

(b) Stainless steel spindle. -™ Fabricate from single contin- 
uous piece of stainless steel wire Vie" diam. and bent to contain 
3 horizontal extensions 5 cm (2") long connected by 2 vertical 
sections ca 5 cm (2") long. Shape so that vertical sections form 
150° angle, and sharpen free ends of 2 outer horizontal exten- 
sions to point (see Fig. 972.04). Use as carrier for test fabric. 
Primary objective of spindle is to prevent wadding or lodging 
of test fabric during agitation in exposures to test chem. solns. 

(c) Exposure chamber. — Clean, dry 1 pt Mason jar with 
rubber washer or gasket and metal screw cap. 

(d) Agitator. — Device to rotate Mason jar thru 360° vertical 
orbit of 10-20 cm (4-8") diam. at 45-60 rpm for 5 min. Laun- 
derometer or Tumble Jar described in AATCC70 B-1967, 43, 
B154, B155, or ASTM D583-63 is adequate. 

(e) Water bath. — Thermostatically controlled at 25°. 

(f) Petri dishes. — Sterile, 100 X 15 mm. 

(g) Glassware.— See 955.11B(a). 

(h) Transfer loops and needles. — See 955.11B(d). 

C. Preparation of Fabric 

(a) Fabric mounting. — Pierce one end of prescoured, 15 g 
test fabric strip and secure onto an outer horizontal extension 
of test spindle; then wind strip around 3 horizontal extensions 
with enough tension to obtain 12 (but not 13) entire laps. Se- 
cure final end of test fabric strip to previous laps with stainless 
steel safety pin. 

(b) Fabric treatment with product. — Dil. product as di- 
rected to 75 mL (most frequently, use directions are based on 
dry wt of laundry fabric equiv. to 15 g test fabric), add to 
Mason jar (exposure chamber), and maintain in H 2 bath at 
25°. Add addnl materials to Mason jar as required by use di- 
rections for product. These are: 

(J) Product recommended as final rinse additive in indus- 
trial laundering operation. — Add no addnl materials; 5:1 
(v/w) treatment product soln to dry fabric ratio is represen- 
tative of industrial laundering operations. 



Outer Extension 




zz* 



Horizontal 
Extensions 



5/16" 



2~ 
o 



Side 

View 



Horizontal 
Extensions 



Outer Extension 



:>* _L 



* Points 
pencil 
sharp or 
canted 



FIG. 972.04 — Stainless steel spindle for winding test fabric 



(2) Product recommended as final rinse additive in home 
or coin-operated laundering operations. — Add 150 mL H 2 
to Mason jar. Resultant 10:1 (v/w) treatment product soln to 
dry fabric ratio is representative of home and coin -operated 
laundering operations. 

(3) Product recommended as final rinse additive in both in- 
dustrial and home laundering operations. — Prep. 2 jars contg 
product soln according to (/) and (2) so that 2 test fabric strips 
may be treated at different treatment product soln to dry fabric 
ratios (5:1 and 10:1 (v/w)). 

(4) Product recommended as final rinse additive and de- 
scribed as compatible with adjunct chemicals which may be 
used in this cycle (sours, bleaches, optical brighteners, sof- 
teners, etc). — Prep, so that required vol. of product treatment 
soln contains adjunct chemicals according to description and 
amts specified on product label or advertising literature. 

D. Operating Technic 

Place test spindle with test fabric in Mason jar contg product 
soln. Secure rubber gasket and Mason jar cap, remove from 
H 2 bath, place jar in agitator, and rotate 5 min. Addnl ma- 
nipulation with test spindle is required if use directions do not 
specify addn of product in final rinse phase of laundry cycle. 
In this instance, to det. durability of antimicrobial agent in 
fabric, execute 3 rinse operations as follows: Immediately after 
end of initial 5 min agitation, drain treatment soln from Mason 
jar and replace with 100 mL H 2 0. Secure Mason jar contg test 
spindle, return to agitator, and rotate 2 min. Repeat operation 
twice more. 

Following all required fabric treatment operations, remove 
test spindle from Mason jar and unwind test fabric strip from 
spindle. Let test fabric strip air dry with long axis of strip in 
horizontal position. 

When test strip is dry, remove 1 sq in. bacteriological test 
samples. Five test samples are required for single bacterio- 
static test against 1 test organism. In each such instance, at 
least 2 test samples must be removed from middle 20% of 
length of test strip. 

Perform bacteriostatic agar plate tests as follows: Prep. 5 
replicate plates in each test for each organism. Sep. inoculate 
flasks contg 100 mL sterile, liq. (<40°) nutrient agar B with 
1 mL 24 hr nutrient broth culture of S. aureus and K. pneu- 
moniae, aberrant. Immediately thereafter, if desired, add 1 mL 
appropriate soln of 2,3,5-triphenyl tetrazolium chloride to in- 
oculated nutrient agar B . Vigorously swirl contents of erlen- 
meyers to ensure complete mixing. Add 10 mL portions of 
inoculated agar to 100 mm sterile petri dishes, distribute evenly, 
and let cool and harden. As soon as plates harden, implant 
single 1 sq in. treated fabric test sample on center of 1 test 
agar plate surface. Using blunt forceps, press each fabric test 
sample onto agar surface to ensure complete and uniform con- 
tact. Incubate test plates 48 hr at 37°. If desired, test plates 
may be refrigerated 18-20 hr before incubation. Following in- 
cubation, examine test plates to det. presence or absence of 
zones of inhibition along each side of test fabric sample. 

E. interpretation 

Use clear zone of inhibition adjacent to each side of test 
fabric sample as index of bacteriostatic activity. Size of zone 
is not considered important, but zone is required to extend along 
entire edge to be acceptable. Score zone of inhibition along 
single side of sq test fabric samples as 1 , so that for 5-replicate 
plate test, a score of 20 shows that bacterio stasis occurs along 
all 4 sides of each sample. Total score of 18/20 sides dem- 
onstrating bacteriostasis is required for effective demonstration 
of residual bacteriostatic activity of laundry fabric treated with 
antimicrobial laundry additive product during laundering op- 
eration. Unless qualified residual bacteriostatic claim is made, 



AOAC Official Methods of Analysis (1990) 



Pool Disinfectants 



145 



residual treatment must be bacteriostatic against both S. aureus 
and K. pneumoniae, aberrant. 

Ref.: JAOAC52, 836(1969). 



965.13 Disinfectants (Water) 

for Swimming Pools 

First Action 1965 
Final Action 1970 

(Suitable for presumptive evidence of acceptability of products 
for disinfecting swimming pool water) 

A. Test Culture Media 

(a) Nutrient Agar A. —See 960.09A(a)(7). 

(b) Nutrient Agar B (Trypticase Soy Agar, BBL). — See 
965.13B(b). 

(c) Nutrient Agar C. — Prep, as in 955.11A(c). 

B. Subculture Media 

(a) Tryptone glucose extract agar (Difco). — Dissolve 24 g 
in 1 L freshly distd H 2 and heat to bp to dissolve completely. 
Autoclave 15 min at 121°. Use for plate counts of E. coli sur- 
vivors. 

(b) Trypticase soy agar (BBL). — Suspend 40 g powder in 
1 L H 2 0. Let stand 5 min and mix thoroly. Heat gently with 
occasional agitation and boil ca 1 min or until soln is com- 
plete. Autoclave 15 min at 121°. Let cool and reautoclave 15 
min at 121°. Use for plate counts of S. faecalis survivors. 

(c) Fluid thioglycolate medium (Difco). — See 955.11A(d)(2). 

(d) Lactose broth (Difco). — Dissolve 19 g in 1 L H 2 t). Dis- 
pense 10 mL portions into tubes with fermentation vials. Au- 
toclave 15 min at 121°. Use for detg presence of E. coli sur- 
vivors. 

(e) Eosin methylene blue agar (Difco). — Suspend 36 g in 
1 L H 2 and heat to bp to dissolve completely. Autoclave 15 
min at 121°. Use for confirming E. coli survivors. 

(f) S-F agar (Difco).— Dissolve 36 g in 1 L H 2 0. Add 15 
g agar and heat to bp to dissolve completely. Autoclave 15 
min at 121°. Use for confirming S. faecalis survivors. 

C. Neutralizer Stock Solns 

(a) Sodium thiosulfate soln. — Dissolve 1 g Na 2 S 2 3 in 1 L 
H 2 0. Dispense in 100 mL portions and autoclave 20 min at 
15 lb. 

(b) Azolectin soln.— See 960.09A(c). 

(c) Other preparations . — Prepns found to be suitable and 
necessary, depending upon nature of germicidal prepns to be 
tested. 

D. Neutralizer Blanks 

(a) With 0.6 ppm residual chlorine or less. — Dil. 10 mL 
neutralizer stock soln, 965.13C(a), with 90 mL sterile H 2 0. 
Dispense aseptically in 9 mL portions into sterile 25 x 150 
mm tubes. 

(b) With quaternary ammonium compounds and phenolic 
derivatives. — Mix 10 mL neutralizer stock soln, 965.13C(b), 
2.5 mL 0.25M phosphate buffer stock soln, 965.13E(a), and 
167.5 mL H 2 0. Dispense in 9 mL portions into 20 X 150 mm 
tubes. Autoclave 20 min at 121°. 

(c) Other preparations. — Use dims of 965.13C(c) as suit- 
able. 

E. Reagents 

(a) Phosphate buffer stock soln.—0.25M. See 960.09A(e). 

(b) Phosphate buffer dilution water. — See 960.09A(f). 

(c) Sodium thiosulfate std solns. — (J) 0.1N. Dissolve ex- 



actly 24.820 g Na 2 S 2 3 .5H 2 in H 2 and dil. to 1 L. Stdze 
as in 942.27B. 

(2) 0.001N.—DW. 10 mL soln (7) to 1 L with H 2 0. 

(d) Starch indicator soln. — Mix ca 2 g finely powd. potato 
starch with cold H 2 to thin paste; add ca 200 mL boiling 
H 2 0, stirring constantly, and immediately discontinue heating. 
Add a few drops of CHC1 3 as preservative. 

(e) Sterile phosphate buffer stock solns. — (7) Dissolve 11.61 
g anhyd. K 2 HP0 4 in 1 L H 2 and autoclave 20 min at 121°. 
(2) Dissolve 9.08 g anhyd. KH 2 P0 4 in 1 L H 2 and autoclave 
20 min at 121°. 

(f) NaOCl stock soln. — Approx. 5%. Store NaOCl stock 
soln in tightly closed bottle in refrigerator and det. exact avail- 
able CI at frequent intervals by As 2 3 titrn, 955.16A(c). 

(g) Test organism. — Use Escherichia coli ATCC 11229 and 
Streptococcus faecalis ATCC No, 6569 (American Type Cul- 
tures Collection, 12301 Parklawn Dr, Rockville, MD 20852). 
Maintain, by monthly transfer, stock cultures of E. coli on 
Nutrient Agar C, 955.11A(c), and 5 '. faecalis on Nutrient Agar 
B, 965.13B(b); store at 4-5°. 

F. Apparatus 

(a) Glassware. — 500 mL wide-mouth erlenmeyers; 100 mL 
graduates; Mohr pipets; milk pipets; 20 x 150 mm tubes; Board 
of Health tubes (6mm x 50mm); 200, 500, and 1000 mL vol. 
flasks. Wash in strong, fresh chromic acid cleaning soln, and 
fill and drain with H 2 >3 times. Heat >2 hr at 180° in hot 
air oven. 

(b) Petri dishes. — Sterile. 

(c) Water bath. — Controlled at 20 or 25°. 

G. Preparation of Culture Suspension 

From stock culture, inoculate tube Nutrient Agar A for E. 
coli and Nutrient Agar B for S. faecalis; make ^3 consecutive 
daily transfers (<30), incubating transfer 20-24 hr at 35-37°. 
Do not transfer >30 days. If only 1 daily transfer has been 
missed, no special procedures are required; if 2 daily transfers 
are missed, repeat with 3 daily transfers. Remove culture from 
agar surface, using 5 mL phosphate buffer diln H 2 0, 
965.13E(b). Transfer culture suspension to sterile centrf. tube 
and centrf. 1-2 min at speed necessary to settle agar particles. 
Transfer supernate to another sterile centrf. tube and centrf. to 
obtain complete sepn of cells. Discard supernate and resus- 
pend cells in 5 mL buffer diln H 2 0. With S. faecalis, centrf., 
discard supernate, and resuspend cells in 5 mL buffer diln H 2 
2 addnl times. Finally, stdze suspension to give av. of 2.0 X 
10 8 organisms/mL by diln with sterile phosphate diln H 2 0. 

If Lumetron is used, dil. suspension in sterile Lumetron tube 
to give % T according to Table 965.13. Make serial diln plate 
count of each culture suspension before use, using phosphate 
buffer diln H 2 0, 965.13E(b), and subculture medium, 
965.13B(a), with E. coli, and (b) with S. faecalis. Incubate 
diln plates in inverted position 48 hr at 35-37°. Use Quebec 
Colony Counting Chamber and report results in terms of num- 
ber of bacteria/mL suspension. Count of 2.0 X 10 8 is desired 
so that 1 mL test culture suspension + 199 mL test soln will 
provide soln contg 1 x 10 6 organisms/mL. Permitted variation 

Table 965.13 Percent Light Transmission at Various Wave- 
lengths Corresponding to Bacterial Concentra- 
tions 







% 


Light Transmission 
with Filter, nm 




Av. 
Bacterial 


Bacteria 


370 


420 


490 530 580 


650 


Count/mL 


E. coli 
S. faecalis 


90 
86 


88 
82 


89 88 91 
85 85 87 


92 
89 


2.0 x 10 8 
2.0 x 10 8 



146 



Disinfectants 



AOAC Official Methods of Analysis (1990) 



in test culture suspension is +500,000 and — 100,000/mL of 
200 mL test soln. Use actual count for calcg zero time count 
in later tests. 

H. Determining Chlorine Demand of Freshly Distilled Test 
Water 

Place 200 mL H 2 in each of five 500 mL erlenmeyers. To 
flasks 1-5, resp., add 0.025, 0.05, 0.075, 0.1, and 0.15 mL 
of 200 ppm available CI prepd from NaOCl soln, 965.13E(f). 
Shake each flask, and let stand several min. Add crystal KI 
and 1 mL HOAc, and swirl. Add 1 mL starch soln, 965.13E(d). 
Flask showing perceptible blue indicates CI demand has been 
satisfied. 

/. Operating Technic 

Place ca 600 mL freshly sterilized distd H 2 in 1 L vol. 
flask. Add ca 1.5-3.0 mL K,HP0 4 buffer, 965.13E(e)(7), and 
0.5 mL KH 2 P0 4 , 965.13E(e)(2), and dil. to 900 mL. Add 
enough NaOCl from suitable diluent of std stock soln, 
965.13E(f), to satisfy CI demand of 1 L test H 2 0, 965.13H, 
and to provide ca 0.6 ppm residual available CI. Dil. to vol. 
(Example: If CI demand of H 2 is 0.1 ppm, add 3.5 mL of 
200 ppm soln of available CI made from std stock NaOCl soln , 
965.13E(f), and dil. to vol. This should provide soln with ca 
0.6 ppm residual available CI at pH 7. 5 ±0.1.) Transfer 199 
mL of this test soln to each of three 500 mL erlenmeyers and 
place in H 2 bath at either 20 or 25°. Let come to temp. 

To first flask, add 1 mL boiled distd H 2 and det. residual 
available CI as follows: Add small crystal KI and 1 mL HOAc; 
then add 1 mL starch soln, 965.13E(d). Blue soln indicates 
presence of CI. Titr. with 0.00W Na 2 S 2 3 , 965.13E(c)(2), 
until color disappears; mL O.OOIA' Na 2 S 2 3 X 0.1773 - ppm 
residual available CI. This represents available CI at time in 
test. Result should be ^0.58 but <0.62. 

To each of remaining flasks add 1 mL test culture suspen- 
sion, 965. 13G, as follows: Swirl flask, stopping just before 
suspension is added, to create enough centrifugal motion to 
prevent pooling of suspension at point of contact with test H 2 0. 
Add suspension midway between center and edge of liq. sur- 
face, immersing tip of pipet slightly below surface of H 2 0. 
Avoid touching pipet to neck or side of test flask during op- 
eration. 

From one of these 2 flasks transfer 1 mL aliquots to neu- 
tralizer blanks, 965.13D(a), after intervals of 0.5, 1, 2, 3, 4, 
5, and 10 min. Shake neutralizer blank thoroly immediately 
after adding sample. Prep, serial diln plate counts from neu- 
tralizer blanks, using phosphate buffer diln H 2 0, 965.13E(b), 
and subculture medium, 965.13B(a) for E. coli, and (b) for 
S. faecalis. 



After prepg diln plate counts, inoculate 5 lactose broth tubes, 
965.13B(d), with 1.0 mL aliquots from each neutralizer blank 
tube for each time interval when E. coli is used as the test 
organism, and 5 thioglycolate broth tubes, 965.13B(c), with 
1 .0 mL aliquots from each neutralizer blank tube for each time 
interval when S. faecalis is test organism. 

Incubate all diln plates in inverted position and subculture 
tubes 48 hr at 37°. Use Quebec Colony Counting Chamber in 
reading diln plates and report results in terms of number of 
surviving bacteria/mL test H 2 0. Absence of colony growth on 
diln plates and absence of growth in all 5 lactose or thiogly- 
colate tubes, as case may be, is necessary to show complete 
kill of test organism. 

Immediately after transferring 10 min interval sample from 
second flask to neutralizer blank tube, remove third flask from 
H 2 bath and det. residual available CI exactly as specified 
for first flask. Results should represent residual available CI 
present at 10 min exposure interval. To be acceptable, concn 
of available CI in this flask should be >0.4 ppm. Results in 
CI control test described above should show complete kill of 
E. coli and S. faecalis within 0.5 min. 

With unknown sample, prep. 2 flasks contg 199 mL each 
of soln at concn recommended or to be studied, using CI de- 
mand-free, unbuffered, freshly distd H 2 previously prepd in 
1 L vol. flask where CI demand, as detd above, has been sat- 
isfied by addn of NaOCl soln. Place flasks in H 2 bath at 20 
or 25°; let come to temp. Inoculate 1 flask with 1 mL std test 
culture suspension of E. coli and other with 1 mL std test cul- 
ture suspension of S '. faecalis . Subculture at exactly same time 
intervals and in same manner used with NaOCl control except 
vary composition of neutralizer blank depending upon nature 
of chem. or mixt. of chems under investigation. For example, 
mixt. of Cl-contg chem. and quaternary NH 4 compd would 
require special neutralizer blank prepd by using both neutral- 
izer stock solns, 965.13C(a) and (b). 

Where no concn of chem. under study has been recom- 
mended and objective of study is to det. concn of unknown 
necessary to provide result equiv. to that obtained with CI con- 
trol std, use series of three or four 500 mL flasks contg 199 
mL of various concns of chem. and 1 mL stdzd culture sus- 
pension with each test organism. Report results as log (number 
of survivors) at each time interval both for CI controls and 
various concns of unknown under test. 

Lowest concn of unknown germicide or germicidal mixt. 
providing results equiv. to those obtained with NaOCl as CI 
std is considered lowest concn which could be expected to pro- 
vide acceptable disinfecting activity in swimming pool water. 

Refs.: JAOAC 47, 540(1964); 48, 640(1965). 



7. Pesticide Formulations 

Associate Chapter Editors: 

Peter D. Bland 
ICI Americas, Inc. 

G. Marshals Gentry 

Florida Dept of Agriculture and Consumer Services 

Edwin R. Jackson 

Mississippi State Chemical Laboratory 

James E. Launer 

Oregon Dept of Agriculture 

David F. Tomksns 

Monsanto Co. 



(Chemical names for pesticides mentioned in this chapter are 
given at end of chapter.) 

(Pesticide ref. stds may be available from the following: All- 
tech Associates, 2051 Waukegan Rd, Deerfield, IL 60015; 
Analabs, Inc.; Applied Science; Chem Service Inc., PO Box 
194, West Chester, PA 19380; RFR Corp., 1 Main St, Hope, 
RI 02831; and Supelco.) 



984.03 



Fertilizer-Pesticide 

Formulation Mixtures 

Sampling Methods 

First Action 1984 



See 929.01 and 969.01. 



920.11 



GENERAL METHODS 

935.06 Sampling 

of Pesticide Formulations 
Procedure 

(Caution: See safety notes on pesticides.) 

Examine shipping cases closely for code numbers, different 
labels, and other pertinent information. Give special attention 
to products subject to deterioration. 

Caution: Use care in sampling and transporting toxic ma- 
terials to avoid personal injury and contamination of trans- 
portation facilities in case of breakage. Avoid mutual contam- 
ination with other products during transportation. 

Mark each sample container according to laboratory require- 
ments. 

(a) Small package retail units. — Take one unopened unit 
(1 lb if dry, 1 pt if liq.), except take min. of 2 units of small 
baits in cake form. Size of sample is governed by composition 
of material and anal, methods. 

(b) Large package dry products (25 lb or more). — Sample 
unopened containers, using trier long enough to reach bottom 
of container by inserting into container at one edge or corner 
and probing diagonally toward opposite edge or corner. Take 
cores by code or batch number. Analyze cores from same code 
or batch number as composite or individually. Clean trier mo- 
ral y after sampling each batch. 

Store samples in air-tight glass, metal, plastic, or cardboard 
containers. 

(c) Large package liquid products (5 gallons or more). — 
Use glass, plastic tubing, or stainless steel trier with plunger, 
or rubber tubing for certain materials. Store samples in glass 
or containers of other noncorrosive material with screw top 
caps lined with Teflon or other- inert material. Plastic con- 
tainers may be used only for carefully selected products. 



Pesticide Formulations 

Preparation of Sample 

Final Action 



Thoroly mix all samples before analysis. Det. H 2 0-sol. As 
on samples as received, without further pulverization or drying. 
In case of lye, NaCN, or KCN, weigh large amts in weighing 
bottles and analyze aliquots of their aq. solns. 



920.12 Moisture in Pesticide Formulations 
Final Action 

(Applicable to Paris green, powd Pb arsenate, Ca arsenate, Mg 

arsenate, Zn arsenite, powd Bordeaux mixt., and Bordeaux 

mixt. with arsenicals) 

Dry 2 g to const wt at 105-110° and report loss in wt as 
moisture. 



922.03 Arsenic (Total) 

in Pesticide Formulations 

Hydrazine Sulfate Distillation Method 

Final Action 

(Nitrates do not interfere. Applicable to detn of total As in 
Paris green, Pb arsenate, Ca arsenate, Zn arsenite, Mg arse- 
nate, and Bordeaux mixt. with arsenicals) 

A. Reagents 

(a) Arsenious oxide std soln. — 0.1 or 0.05/V. See 939.12. 

(b) Iodine std soln.— -0. 1 or 0.05/V. See 939.13. 

(c) Bromate std soln. — 0.1 or 0.05iV. Dissolve ca 2.8 or 
1.4 g KBr0 3 in boiled H 2 and dil. to 1 L. Stdze as follows: 
Pipet 25 mL aliquots As 2 3 soln, (a), into 500 mL erlenmey- 



147 



148 



Pesticide Formulations 



AOAC Official Methods of Analysis (1990) 



ers. Add 15 mL HC1, dil. to 100 mL, heat to 90°, and titr. 
with the KBr0 3 soln, using 10 drops Me orange, (g). Do not 
add indicator until near end of titrn, and agitate soln contin- 
uously to avoid local excess of KBr0 3 soln. Add KBr0 3 soln 
very slowly near end point; at end point soln changes from red 
to colorless. 

(d) Hydrazine sulfate- sodium bromide soln. — Dissolve 20 
g N 2 H 4 .H 2 S0 4 and 20 g NaBr in 1 L HC1 (1+4). 

(e) Sodium hydroxide soln. — Dissolve 400 g NaOH in H 2 
and dil. to 1 L. 

(f) Starch indicator. — Mix ca 2 g finely powd. potato starch 
with cold H 2 to thin paste; add ca 200 mL boiling H 2 0, stir- 
ring constantly, and immediately discontinue heating. Add ca 
1 mL Hg, shake, and let soln stand over the Hg. 

(g) Methyl orange indicator. — 0.05%. Dissolve 0.5 g Me 
orange in H 2 and dil. to 1 L. 

B. Apparatus 

See Fig. 922.03. Set 500 mL distn flask on metal gauze that 
fits over circular hole in heavy sheet of asbestos board, which 
in turn extends out far enough to protect sides of flask from 
direct flame of burner. First receiving flask holds 500 mL and 
contains 40 mL H 2 0; second holds 500 mL and contains 100 
mL H 2 0. Vol. in first flask should be <40 mL, otherwise compd 
of As may sep. that is difficult to dissolve without danger of 
loss of AsCJ 3 . Keep both flasks cool by placing in pan of cir- 
culating H 2 0, or contg H 2 and ice. 

C. Determination 

(Caution: See safety notes on pesticides and arsenic trioxide.) 

Weigh sample contg ^0.4 g As and transfer to distg flask. 
Add 50 mL N 2 H 4 .H 2 S0 4 -NaBr soln, close flask with stopper 
that carries funnel tube, and connect side tube with condenser. 
Boil 2-3 min, add 100 mL HC1 from dropping funnel, and 
distil until vol. in distg flask is reduced to ca 40 mL; add 50 
mL more HC1 and continue distn until vol. is again reduced 
to ca 40 mL. Wash down condenser, transfer contents of re- 
ceiving flasks to 1 L vol. flask, dil. to vol., mix thoroly, and 
proceed as in (a) or (b): 

(a) Pipet 200 mL aliquot into erlenmeyer and nearly neutze 
with NaOH soln, using few drops phthln, and keeping soln 
well cooled. If neut. point is passed, add HO until again slightly 
acid. Neutze with NaHC0 3 , add 4-5 g excess, and add std I 
soln from buret, shaking flask continuously until yellow color 



disappears slowly from soln. Add 5 mL starch indicator and 
keep adding std I soln dropwise to permanent blue. 

(b) Pipet 200 mL aliquot into erlenmeyer and titr. with KBr0 3 
soln, (c), beginning "... heat to 90° ..." 

Calc. % As. Report as As 2 3 or As 2 5 , according to whether 
As is present in trivalent or pentavalent form. If condition of 
arsenic is unknown, report as As. 

Refs.: Ind. Eng. Chem. 14, 207(1922). JAOAC 5, 33, 
402(1922); 6, 313(1923); 48, 564(1965). 

CAS-7440-38-2 (arsenic) 

924.04 Arsenic (Total) 

in Pesticide Formulations 

lodometric Method 

Final Action 

(Applicable in presence of sulfides, sulfites, thiosulfates , and 
large amts of S or org, matter) 

A. Reagent 

Sodium thiosulfate soln. — Dissolve 13 g crystd Na 2 S 2 3 .5H 2 
in H 2 and dil. to 1 L. 

See 922. 03 A for other reagents and solns and 922. 03B for 
app. 

B. Determination 

(Caution: See safety notes on pesticides and arsenic tri- 
oxides.) 

Weigh sample contg <0.4g As and transfer to distg flask. 
Add 50 mL N 2 H 4 .H 2 S0 4 -NaBr soln, 922.03A(d), and distil as 
in 922.03C. Dil. distillate to vol. in 1 L vol. flask, mix tho- 
roly, and transfer 200 mL aliquot to 400 mL Pyrex beaker or 
porcelain casserole. Add 10 mL HN0 3 and 5 mL H 2 S0 4 , evap. 
to sirupy consistency on steam bath, and then heat on hot plate 
to white fumes of H 2 S0 4 . Cool, and wash into 500 mL erlen- 
meyer. If vol. H 2 S0 4 is appreciably lessened by fuming, add 
enough H 2 S0 4 to make total vol. ca 5 mL. Dil. to 100-150 
mL, add 1.5 g KI, and boil until vol. is reduced to ca 40 mL. 
Cool under running H 2 0, dil. to 100-150 mL, and add Na 2 S 2 3 
soln, 924. 04A, dropwise until I color just disappears. Nearly 
neutze H 2 S0 4 with NaOH soln, 922.03A(e), finish neutzn with 
NaHC0 3 , add 4-5 g excess, and titr. with std I soln as in 




FIG. 922.03 — Apparatus for distilling arsenious chloride 



AOAC Official Methods of Analysis (1990) 



General Methods 



149 



922.03C(a). From mL std soln used, calc. % As in sample. 
Report as As 2 3 , As 2 O s , or As as in 922. 03C. 

Ref.: JAOAC 7, 313(1924). 

CAS-7440-38-2 (arsenic) 



963.06 Arsenic (Total) 

in Pesticide Formulations 
Bon Exchange Method 

First Action 1963 
Final Action 1968 

(Applicable to inorg. arsenates and arsenites) 

A. Apparatus 

Ion exchange column. — Use All inn filter tube 10 X 2.7 cm 
od with coarse filter disk. Attach piece of rubber tubing to 
bottom of filter tube and regulate flow with Hoffman clamp. 
To tube add aq. slurry of Dowex 50W-X8, 50-100 mesh, us- 
ing resin bed vol. of 12 mL, and place 500 mL separator above 
tube. 

Regenerate resin bed before each run by first back-washing 
column few min with H 2 0; then elute with 350 mL IN HC1 
followed by 200 mL H 2 6 at 20 mL/min. 

B. Preparation of Sample 

(Caution: See safety notes on pesticides.) 

Weigh 200 mg sample (100 mg if As content is >30%) into 
150 mL beaker, add 7 mL HN0 3 , and bring to bp. Add 3 mL 
2N KBr0 3 and evap. to dryness, avoiding spattering. Back- 
wash and regenerate resin during this evapn. Dissolve cooled 
residue in 2 mL 67V HC1 without heat and add 8 mL H 2 0. Filter 
into separator, and wash filter with three 10 mL portions H 2 0. 
(If residue dissolves completely in 2 mL 6/V HO, omit filtra- 
tion, and dil. directly to 40 mL.) Pass soln thru resin column 
at 20 mL/min and collect eluate in 250 mL erlenmeyer. Wash 
separator and column with 20 and 40 mL portions H 2 into 
same erlenmeyer. 

C. Determination 

Add 50 mL HC1 to eluate to make 4/V. Add 1 g NaHC0 3 , 
0.2 g at time, swirling constantly. Add 1 g KI, stopper, and 
swirl until all KI dissolves. After 5 min, titr., without starch 
indicator, with 0.05N Na 2 S 2 3 , 942.27, to disappearance of I. 
(Recognition of end point is facilitated by titrg on porcelain 
stand. In presence of starch, reaction between I and Na 2 S 2 3 
is retarded, so appreciable amt of Na 2 S 2 3 reacts with acid. 
End point becomes indistinct if >30 mL Na 2 S 2 3 is used in 
titrn.) 1 mL 0.05A^ Na 2 S 2 3 = 1.873 mg As. 

Refs.: Anal. Chem. 22, 1066(1950). JAOAC 46, 672(1963). 

CAS-7440-38-2 (arsenic) 



925.02 Arsenic (Water-Soluble) in 

Pesticide Formulations 

Titrimetric Method 
Final Action 



to 32°. Stopper flask and place in constant temp. H 2 bath at 
32°. Digest 24 hr, shaking hourly 8 hr during this period. Filter 
thru dry filter. If filtrate is not clear, refilter thru buchner contg 
paper and enough Filter-Cel coating to give clear soln. Discard 
first 50 mL. 

Transfer 250-500 mL clear filtrate to erlenmeyer, add 3 mL 
H 2 S0 4 , and evap. to ca 100 mL on hot plate. Add 1 g KI, and 
continue boiling until vol. is ca 40 mL. Cool, dil. to ca 200 
mL, and add Na 2 S 2 3 soln, 924. 04A, dropwise, until I color 
is exactly removed. (Avoid use of starch indicator at this point.) 
Neutze with NaHC0 3 , add 4-5 g excess, titr. with std I soln, 
shaking flask continuously, until yellow disappears slowly, add 
5 mL starch indicator, 922.03A(f), and continue titrn to per- 
manent blue. Correct for amt std I soln necessary to produce 
same color, using same reagents and vol. From mL std I soln 
used, calc. % H 2 0-sol. As in sample. 

CAS-7440-38-2 (arsenic) 



922.04 Lead in Pesticide Formulations 

Gravimetric Method 
Final Action 

(Applicable to such prepns as Bordeaux-Pb arsenate, Bor- 
deaux-Zn arsenite, Bordeaux-Paris green, and Bordeaux-Ca 

arsenate) 

(Caution: See safety notes on nitric acid, fuming acids, pes- 
ticides, hydrogen sulfide, and arsenic trioxide.) 

Weigh 1 g powd sample and transfer to beaker. Add 5 mL 
HBr (ca 1.38 sp gr) and 15 mL HO, and evap. to dryness to 
remove As. Repeat treatment; add 20 mL HO, and again evap. 
to dryness. Add 25 mL 2N HO to residue, heat to bp, filter 
immediately to remove Si0 2 , and wash with boiling H 2 to 
vol. of 125 mL. See that all PbCl 2 is in soln before filtering; 
if it will not dissolve completely in 25 mL IN acid, add 25 
mL more and dil. filtrate to 250 mL. Pass in H 2 S until pptn 
is complete. Filter, and wash ppt thoroly with 0.5 A" HO satd 
with H 2 S. Save filtrate and washings for Zn detn. 

Transfer paper with sulfides of Pb and Cu to 400 mL Pyrex 
beaker and completely oxidize all org. matter by heating on 
steam bath with 4 mL H 2 S0 4 and ca 20 mL fuming HNO3 in 
covered beaker. Evap. on steam bath, and then completely re- 
move HN0 3 by heating on hot plate to copious white fumes 
of H 2 S0 4 . Cool, add 2-3 mL H 2 0, and again heat to fuming. 
Cool, add 50 mL H 2 and 100 mL alcohol, and let stand sev- 
eral hr (preferably overnight). Filter thru gooch, previously 
washed with H 2 0, then with acidified alcohol (100 parts H 2 0, 
200 parts alcohol, and 3 parts H 2 S0 4 ), and finally with alco- 
hol, and dried at 200°. Wash ppt of PbS0 4 in crucible ca 10 
times with acidified alcohol, and then with alcohol, to remove 
H 2 S0 4 . Retain filtrate and washings for Cu detn, if desired. 

Dry at 200° to const wt, keeping crucible covered to prevent 
loss from spattering. From wt PbS0 4 , calc. % Pb in sample, 
using factor 0.6832. 

Ref.: JAOAC 5, 398(1922). 

CAS-7439-92-1 (lead) 



(Applicable to detn of H 2 0-sol. arsenic in Pb arsenate, Ca ar- 
senate, Zn arsenite, Mg arsenate, and Bordeaux mixt. with 
arsenicals) 

(Caution: See safety notes on pesticides.) 

To 2 g original sample if powder, or 4 g if paste, in 1 L 
Florence flask, add 1 L recently boiled H 2 that has been cooled 



922.05 Copper in Pesticide Formulations 
Final Action 

(Applicable to such prepns as Bordeaux-Pb arsenate, Bor- 
deaux-Zn arsenite, Bordeaux-Paris green, and Bordeaux-Ca 

arsenate) 



150 



Pesticide Formulations 



AOAC Official Methods of Analysis (1990) 



A. Electrolytic Method 

Evap. filtrate and washings from PbS0 4 pptn, 922.04, to 
fuming; add few mL fuming HN0 3 to destroy org. matter, and 
continue evapn to ca 3 mL. Take up with ca 150 mL H 2 0, 
add 5 mL HN0 3 , and filter if necessary. Wash into 250 mL 
beaker, adjust vol. to 200 mL, and electrolyze, using rotating 
anode and weighed gauze cathode with current of 2-3 amp. 
After all Cu has apparently deposited (ca 30 min), add 15-20 
mL H 2 to electrolyte and continue electrolysis few min. If 
no further deposition occurs on newly exposed surface of elec- 
trode, wash with H 2 without breaking current either by si- 
phoning or quickly replacing beaker with electrolyte succes- 
sively with 2 beakers of H 2 0. Interrupt current, rinse cathode 
with alcohol, dry few moments in oven, and weigh. Calc. % 
Cu in sample. 

B. Volumetric Thiosulfate Method 

Proceed as in 922. 05A to point at which filtrate and wash- 
ings from PbS0 4 pptn are treated with fuming HN0 3 and evapd 
to vol. of ca 3 ml, Take up in ca 50 mL H 2 0, add NH 4 OH 
in excess, and boil to expel excess NH 3 , as shown by color 
change in liq. and partial pptn. Add 3-4 mL HOAc (4+1), 
boil 1-2 min, cool, add 10 mL 30% KI soln, and titr. with 
std Na 2 S 2 3 soln until brown color becomes faint. Add starch 
indicator, 922.03 A(f), and continue titrn cautiously until blue 
color due to free I entirely disappears. From mL std Na 2 S 2 3 
soln used, calc. % Cu in sample. 

Thiosulfate std soln. — Prep, soln contg 39 g Na 2 S 2 3 .5H 2 0/ 
L. Accurately weigh 0.2-0.4 g pure electrolytic Cu and trans- 
fer to 250 mL erlenmeyer roughly marked at 20 mL intervals. 
Dissolve Cu in 5 mL HN0 3 (1 + 1), dil. to 20 or 30 mL, boil 
to expel red fumes, add slight excess satd Br-H 2 0, and boil 
until Br is completely removed. Cool, and add 10 mL NaOAc 
soln (574 g trihydrate/L). Prep. 42 g/100 mL KI soln made 
very slightly aJk. to avoid formation and oxidn of HI. Add 10 
mL of the KI soln and titr. with Na 2 S 2 3 soln to light yellow. 
Add enough starch indicator, 922. 03 A (f), to produce marked 
blue. As end point nears, add 2 g KSCN and stir until com- 
pletely dissolved. Continue titrn until ppt is perfectly white. 1 
mL Na 2 S 2 3 soln = ca 10 mg Cu. 

It is essential for Na 2 S 2 3 titrn that concn of KI in soln be 
carefully regulated. If soln contains <320 mg Cu, at comple- 
tion of titrn 4.2-5 g KI should have been added for each 100 
mL total soln. If greater amts of Cu are present, add KL soln 
slowly from buret with const agitation in amts proportionately 
greater. 

Ref.: JAOAC 5, 398(1922). 

CAS-7440-50-8 (copper) 



918.01 Zinc in Pesticide Formulations 

Gravimetric Method 
Final Action 

(Applicable to such prepns as Bordeaux-Pb arsenate, Zn ar- 

senite, Bordeaux-Zn arsenite, Bordeaux-Paris green, and Bor- 

deaux-Ca arsenate) 

A. Reagent 

Mercury-thiocyanate soln. — {Caution: See safety notes on 
mercury salts.) Dissolve 27 g HgCl 2 and 30 g NH 4 SCN in H 2 
and dil. to 1 L. 

B. Determination 

Cone, filtrate and washings from sulfide pptn, 922.04, by 
gentle boiling to ca 50 mL; then evap. on steam bath to dry- 



ness. Dissolve residue in 100 mL H 2 contg 5 mL HC1, and 
add 35-40 mL Hg-thiocyanate soln with vigorous stirring. Let 
stand ^1 hr with occasional stirring. Filter thru weighed gooch, 
wash with H 2 contg 20 mL Hg-thiocyanate soln/L, and dry 
to const wt at 105°. Calc. to % Zn, using factor 0.1312. 

Note: Some Fe is usually present and during Zn detn should 
be in ferrous condition. In pptg sulfides pass H 2 S into soln 
long enough to reduce Fe as well as to ppt Cu and Pb. 
ZnHg(SCN) 4 ppt normally is white, and occluded Fe(SCN) 3 
should not give more than faint pink color. 

Refs.: J. Am. Chem. Soc. 40, 1036(1918). JAOAC 5, 
398(1922). 

CAS -7440-66-6 (zinc) 



929.04 Fluorine (Total) 

in Pesticide Formulations 

Lead Chlorofluoride Method 

Final Action 

A. Reagents 

(a) Fusion mixture. — Mix 30 g anhyd. Na 2 C0 3 with 40 g 
anhyd. K 2 C0 3 . 

(b) Lead chlorofluoride wash soln. — Dissolve 10 g Pb(N0 3 ) 2 
in 200 mL H 2 0, dissolve 1 g NaF in 100 mL H 2 and add 2 
mL HO, and mix these 2 solns. Let ppt settle and decant. 
Wash ppt 4 or 5 times with 200 mL H 2 by decanting; then 
add ca 1 L cold H 2 to ppt and let stand >1 hr, with oc- 
casional stirring. Filter and use clear filtrate. (Prep, more 
wash soln as needed by adding more H 2 to ppt of PbCIF 
and stirring.) 

(c) Silver nitrate std soln. — 0. 1 or 0.2yV. Stdze as in 941. 18E. 

(d) Potassium or ammonium thiocyanate std soln. — 0.1 jV. 
Stdze against std AgN0 3 soln under same conditions as in detn. 

(e) Ferric indicator. — To cold satd Cl-free 
FeNH4(SO4)2.12H 2 soln add enough colorless H.NO3 to bleach 
brown color. 

(f) Bromophenol blue indicator. — Grind 0. 1 g powder with 
1.5 mL 0.17V NaOH and dil. to 25 mL. 

B. Determination 

(a) Samples difficult to decompose such as cryolite , and 
others that contain aluminum or appreciable amounts of sili- 
ceous material. — Mix 0.5 g sample (or less if necessary to 
contain 0.01-0.10 g F) with 5 g fusion mixt. and 0.2-0.3 g 
powd. Si0 2 in Pt dish, cover with 1 g fusion mixt., and heat 
to fusion over Bunsen burner. (Use of blast lamp is unnec- 
essary since it is preferable not to heat much beyond melting 
temp. If much Al is present, uniform, clear, liq. melt cannot 
be obtained; particles of white solid will sep. in melt. Cooled 
melt should be colorless, or at least should not have more than 
gray color.) 

Leach cooled melt with hot H 2 and when disintegration is 
complete, filter into 400 mL beaker. Return insol. residue to 
Pt dish with jet of H 2 0, add 1 g Na 2 C0 3 , dil. to 30-50 mL, 
and boil few min, disintegrating any lumps with flat-end rod. 
Filter thru same paper, wash thoroly with hot H 2 0, and adjust 
vol. of filtrate and washings to ca 200 mL. Add 1 g ZnO dis- 
solved in 20 mL HN0 3 (1+9), boil 2 min, stirring constantly, 
filter, and wash thoroly with hot H 2 0. During this washing 
return gelatinous mass to beaker 3 times and thoroly disinte- 
grate in wash soln because proper washing of this ppt on filter 
is difficult. (Mass can easily be returned to beaker by rotating 
funnel above beaker while cutting ppt loose from paper with 
jet of wash soln.) 



AOAC Official Methods of Analysis (1990) 



General Methods 



151 



Add 2 drops bromophenol blue to filtrate, and with cover 
glass almost entirely over beaker, add HN0 3 (1+4) until color 
just changes to yellow. Make soln slightly alk. with 10% NaOH 
soln, and with cover glass on beaker, boil gently to expel C0 2 . 
Remove from burner; add the HN0 3 until color just changes 
to yellow and then 10% NaOH until color just changes to blue; 
then add 3 ml 10% NaCl soln. (Vol. of soln at this point 
should be ca 250 mL.) 

Add 2 mL HCI (1 + 1) and 5 g Pb(N0 3 ) 2 and heat on steam 
bath. As soon as Pb(N0 3 ) 2 is in soln, add 5 g NaOAc, stir 
vigorously, and digest on steam bath 30 min with occasional 
stirring. Let stand overnight, filter, and wash ppt, beaker, and 
paper once with cold H 2 0, then 4 or 5 times with PbCIF wash 
soln, and then once more with cold H 2 0. 

Transfer ppt and paper to beaker in which pptn was made, 
stir paper to pulp, add 100 mL HN0 3 (5 + 95), and heat on 
steam bath until ppt dissolves. (5 min is ample to dissolve ppt. 
If sample contains appreciable amt of sulfates, ppt will contain 
PbS0 4 , which will not dissolve. In such case heat 5-10 min 
with stirring and consider PbCIF to be dissolved.) Add slight 
excess 0.LV or 0.27V AgN0 3 , digest on steam bath 30 min, 
and cool to room temp., protecting from light; filter, wash 
with cold H 2 0, and det. AgN0 3 in filtrate by titrn with std 
thiocyanate soln, using 10 mL ferric indicator. Subtract amt 
of AgN0 3 found in filtrate from that originally added. Differ- 
ence is amt required to combine with CI in the PbCIF; from 
this difference calc. % F in sample. 1 mL O.liV AgN0 3 — 
0.00190 g F. 

(b) Water-soluble fluorides in presence of organic mat- 
ter. — In presence of <50% org. matter such as flour, pyreth- 
rum, tobacco powder, and derris or cube powders, which readily 
decompose without addn of powd Si0 2 and contain little or no 
sulfates, Al, or siliceous compds, mix 0.5 g sample (or less 
if necessary to contain 0.01-0.1 g F) with 5 g fusion mixt., 
cover with 1 g fusion mixt,, and heat to fusion over Bunsen 
burner. Leach cooled melt with hot H 2 0, and when disinte- 
gration is complete, filter into 600 mL beaker. Wash thoroly 
with hot H 2 and proceed as in (a), third par. 

In presence of >50% org. matter or org. matter that is im- 
practical to free without preliminary ashing, such as apple peel 
and pulp, transfer enough sample to Pt crucible to be repre- 
sentative of mixt. and to contain 0.01-0.1 g F. Add 15 mL 
H 2 and enough F-free CaO (0.3-0.4 g) to make mixt. dis- 
tinctly alk. to phthln, mix with glass rod, and evap. to dryness 
on steam bath and in oven at 105°. Ignite at low heat, pref- 
erably in furnace (<600°), until org. matter is thoroly charred. 
Pulverize, with glass rod, any lumps present in charred ash, 
mix with 5 g of the fusion mixt., and proceed as in (a), first 
par., beginning "... cover with 1 g fusion mixt., ..." 

(c) Water-soluble samples in absence of organic matter and 
appreciable quantities of sulfates or aluminum salts. — In ab- 
sence of org. matter or other interfering substances, fusion may 
be omitted and detn made on aliquot of aq. soln contg 0.01- 
0.1 g F, as in (a), third par. 

In presence of Al, as in samples contg Na 2 SiF 6 and 
KAI(S0 4 ) 2 1 2H 2 0, transfer sample to 400 mL beaker, dissolve 
in 150 mL hot H 2 0, add 6 g fusion mixt., and boil. Add 1 g 
ZnO dissolved in 20 mL HNO3 (1 + 9), boil 2 min with const 
stirring, filter into 500 mL vol. flask, and wash thoroly with 
hot H 2 0. Cool to room temp, and dil. to vol. Transfer 200 
mL aliquot contg 0.01-0.10 g F to 600 mL beaker and pro- 
ceed as in (a), third par. 

(d) Sodium and magnesium fluosilicates, or samples con- 
taining more than 5% sulfates in absence of aluminum and 
boron, with or without moderate amounts of organic mat- 
ter. — With large amts of Na 2 SiF 6 and some other more volatile 



fluosilicates, e.g., MgSiF 6 , where there is possibility of some 
F being evolved as SiF 4 before fusion is effected, or in samples 
contg appreciable amts of sulfates, distil F as in 933. 03B, and 
det. F in distillate as follows: Add several drops bromophenol 
blue, make alk. with NaOH, and adjust vol. to ca 250 mL by 
gently boiling down vol. from 400 to 250 mL. Proceed as in 
(a), third par., beginning "Remove from burner; ..." 

Notes: These methods give accurate results for 0.01-0.10 g 
F. Below 0.01 g, results tend to be slightly low, and above 
0.10 g, slightly high. Convenient sample to fuse is one contg 
0.07-0.08 g F; too large sample may result in incomplete fu- 
sion. Large amts of B compds and alkali salts retard or prevent 
complete pptn of PbCIF. B has greater effect when amt of F 
is large than when it is small . In methods described B has little 
effect, and it may be disregarded in analysis of insecticides if 
amt of F to be pptd is <0.03 g. With some prepns contg Na 2 B 4 ? 
or H 3 B0 3 , where it is difficult to obtain representative mixt. 
when extremely small sample (0.1 g) is used for analysis, take 
larger sample and ppt PbCIF from aliquot of fusion soln. Amt 
of alkali carbonates specified in fusion and in washing of insol. 
residue is not large enough to cause low results. If sample 
contains S, remove it with CS 2 and det. F on air-dried residue, 
allowing in calcns for % S removed. {Caution: See safety notes 
on flammable solvents, toxic solvents, and carbon disulfide.) 

Refs.: J. Res. Natl. Bur. Standards 3, 581(1929). JAOAC 25, 
670(1942); 27, 74(1944); 28, 72(1945). 

CAS-7782-41-4 (fluorine) 



921.04 Fluorine (Total) 

in Pesticide Formulations 

Modified Travers Method 

Final Action 

(Applicable in absence of B, Al, and large amts of pyrethrum 
powder) 

(Caution: See safety notes on asbestos.) 

A. Reagents 

(a) Alcoholic potassium chloride soln. — Dissolve 60 g KCI 
in 400 mL H 2 0, add 400 mL alcohol, and test with phthln; if 
soln is not neut., adjust to exact neutrality with NaOH or HCI 
soln. 

(b) Sodium hydroxide std soln. — 0.2/V. Prep, and stdze as 
in 936.16. 

B. Determination 

Treat 0.5 g sample in small beaker with 20-25 mL H 2 0. 
Add 0.3 g finely divided pptd Si0 2 and few drops Me orange. 
Add HCI dropwise until soln assumes apparently permanent 
pink; then add 2 mL excess, cover beaker with watch glass, 
and boil 1 min. Cool to room temp., add 4 g KCI, and stir 
until KCI dissolves. Add 25 mL alcohol and let stand 1 hr, 
stirring frequently. Filter thru gooch contg disk of filter paper 
covered with medium pad of asbestos. Wash ppt with ale. KCI 
soln until one washing does not destroy color made by 1 drop 
0.2N NaOH and phthln (usually 3-4 washings). Transfer cru- 
cible and contents to 400 mL beaker, add 100 mL recently 
boiled H 2 and 1-2 mL phthln, heat, and titr. with std NaOH 
soln. Finish titrn with the F soln actively boiling. Calc. % F. 
1 mL 0.2N NaOH = 0.0057 g F. 

Refs.: Compt. rend. 173, 714, 836(1921). JAOAC 14, 
253(1931). 

CAS-7782-41-4 (fluorine) 



152 



Pesticide Formulations 



AOAC Official Methods of Analysis (1990) 



933.03 Fluorine (Total) 

in Pesticide Formulations 

Distillation Method 

Final Action 

(Applicable to H 2 0-sol. or FLO-insol. insecticides 
in absence of gelatinous Si0 2 , B, and Al) 

A. Reagents 

(a) Sodium alizarin sulfonate indicator.— Dissolve 0.1 g Na 
alizarin sulfonate in 200 mL H 2 0. 

(b) Thorium nitrate soln. — Approx. 0.057V. Stdze in terms 
of g F/mL by titrg F obtained by distn from std NaF as in 
933. 03B. In stdzg for use with 933.036(b), add 5 mL satd 
KMn0 4 soln in addn to other reagents in distn flask. 

B. Determination 

(a) In absence of organic matter. — Weigh sample contg ca 
0.09 g F, and with aid of little H 2 transfer to 250 mL Claisen 
distn flask (B) contg 12 glass beads. Adjust to ca 30 mL and 
close flask with 2-hole rubber stopper, thru which pass ther- 
mometer (D) and 4 mm glass tube, both of which extend into 
soln. (The 4 mm glass tube extends ca 5 cm above rubber 
stopper and by means of rubber tube, E, connects still with 1 
L Florence flask (A) contg H 2 for steam generation. Flask is 
equipped with steam discharge, H, and pressure tube, G. See 
Fig. 933.03.) 

Bring H 2 in steam generating flask to boil with pinchcock, 
F, in release tube open. Connect distg flask to condenser, C, 
and add 25 mL H 2 S0 4 thru top of 4 mm tube, using pipet or 
special funnel. With pinchcock, F, open, connect rubber tub- 
ing to 4 mm tube. Light burner under Claisen flask. Regulate 
flow of steam by adjusting burner flames and pinchcock, F, 
so that vol. of soln is held const and temp, in flask, B, is kept 
at 145-150°. Continue distn until 400 mL distillate collects. 
Dil. to 500 mL in vol. flask, transfer 50 mL aliquot to tall- 
form 150 mL beaker, and add 5 drops indicator, 933.03A(a). 
Adjust acidity with 1% NaOH soln and HC1 (1+249) until 
pink just disappears. Add 2 mL of the HCl, and titr. with 0.057V 




FIG. 933.03— Apparatus for determining fluorine 



Th(N0 3 ) 4 to permanent pink, using buret graduated in 0.05 
mL. 

(b) In presence of organic matter. — (Caution: See safety 
notes on permanganates.) In presence of moderate amts of org. 
matter, transfer sample contg ca 0.09 g F and contg ^0.2 g 
org. matter, with aid of little H 2 0, to 250 mL Claisen distn 
flask contg 12 glass beads. Add 5 mL satd KMn0 4 soln, adjust 
to ca 30 mL, and proceed as in (a), beginning "... close flask 
with 2-hole rubber stopper, ..." 

In presence of large amts of org. matter, transfer sample to 
medium-size Pt dish, add 15 mL H 2 and enough F-free CaO 
to make mixt. distinctly alk. to phthln, mix with glass rod, 
and evap. to dryness on steam bath and in oven at 105°. Ignite 
at low heat, preferably in furnace (<600°), until org. matter 
is thoroly charred. Pulverize any lumps present in charred ash 
with glass rod, transfer to 250 mL Claisen distn flask by brush- 
ing, and finally wash out dish with 30 mL H. 2 S0 4 (1+9). Ex- 
cept to add 22 mL instead of 25 mL H 2 S0 4 , proceed as in (a), 
par. 2. 

Note: If coating of pptd Si0 2 forms on inside of distn flask, 
remove by treatment with hot coned alkali soln, as it is capable 
of retaining F during distn of some samples and giving it up, 
at least in part, in later distns. 

Refs.: J. Am. Chem. Soc. 55, 1741(1933). Ind. Eng. Chem. 
Anal. Ed. 5, 7(1933); 9, 551(1937); 11, 21(1939). 
JAOAC 21, 459(1938); 53, 378(1970). 

CAS-7782-41-4 (fluorine) 



945.05 Fluorine Present 

as Sodium Fluosilicate 

in Pesticide Formulations 

Final Action 

(B, CaO, and alum absent) 

A. Reagents 

Alcoholic potassium chloride and sodium carbonate soln. — 
Dissolve 1 .0 g Na 2 C0 3 in 100 mL ale. KC1 reagent, 921.04A(a). 
For other reagent see 921. 04A. 

B. Determination 

Weigh 1 g sample into Pt dish, and add rapidly, with con- 
tinuous stirring, 50 mL of the ale. KC1 -Na 2 C03 reagent. Do 
not let soln become acid, and if necessary, use more reagent 
to insure alky. 

Continue stirring until all sol. portions of sample dissolve. 
Proceed as in 921. 04B, beginning: "Filter thru gooch ..." 
Calc. % Na 2 SiF 6 (1 mL 0.2N NaOH = 0.009403 g Na 2 SiF 6 ). 

CAS-16893-85-9 (sodium fluosilicate) 



972.05 Organochlorine Pesticide 

Contamination of Pesticide Formulations 
Thin Layer Chromatographic Method 

First Action 1972 
Final Action 1977 

AOAC-CIPAC Method 

(Applicable to detection of contamination by 0.01% chlori- 
nated hydrocarbons such as aldrin, DDT, dieldrin, and endrin, 
and 0.05-0.10% of chlordane, strobane, and toxaphene) 

A. Apparatus 

(a) Thin layer chromatographic apparatus. — See 970. 52F. 

(b) Ultraviolet apparatus. — Sterilamp G-15T8, or equiv. 



AOAC Official Methods of Analysis (1 990) 



Inorganics and Organometallics 



153 



B. Reagents 

(a) Adsorbent. — Aluminum oxide G, Type E (Brinkmann 
Instruments, Inc.). 

(b) Mobile solvents.— (1) n-Hexane, (2) n - he xane- acetone 
(98 + 2), and (3) n-hexane-alcohol (98+2). 

(c) Pesticide std solns. — 1 (Jtg/fxL EtOAc, acetone, or any 
convenient solv. 

C. Preparation of Sample 

Ext 8 g sample with 20 mL acetone in 250 mL erlenmeyer 
by shaking intermittently 5 min. Let solids settle. If soln is 
turbid, filter or centrf. to obtain clear supernate for spotting. 
For samples contg large amts S, use 8 g sample and 20 mL 
pet ether. 

D. Preparation of Piates 

Dissolve 0.1-0.15 g AgN0 3 in 1-2 mL H 2 in 100 mL 
beaker, add 58 mL MeOH, and mix. Weigh 40 g adsorbent, 
(a), in 250 mL flask, add AgN0 3 -MeOH soln, and shake vig- 
orously 20 sec. Apply slurry as 0.25 mm thick layer to five 
20 X 20 cm (8 x 8") plates positioned on plastic mounting 
board. After plates appear dry, store in desiccator over des- 
iccant. When plate is dry, scrape 1 cm strip from side edges 
to ensure even solv. front. Use plate immediately after re- 
moval from desiccator. 

E. Detection 

Pour tt-hexane into glass chromatgc tank to depth of 10-20 
mm. Place 2 paper blotters (ca 7.5 X 22 cm) on each side of 
tank or large blotter covering back of tank and let equilibrate 
>2 hr before use. 

Spot 10 jxL sample ext on plate with 100 uJL syringe. Do 
not disturb adsorbent layer. Also spot std solns of pesticides 
declared as part of formulation. Spots should be ^6 mm diam. 
and placed <30 mm from bottom of plate. Place plate in chro- 
matgc tank, and let plate develop >10 cm. Remove plate and 
expose to shortwave UV, 972.05A(b). (Caution: See safety 
notes on hazardous radiations.) Chlorinated org. pesticides 
should be visible as dark spots against white or light gray 
background. Expose plates >1 hr. Longer exposure will not 
harm plates. 

To confirm identification of pesticide, repeat TLC step with 
different mobile solv., 972.058(b)(2) or (J). 

Ref.: J AOAC 55, 851(1972). 



960.10 Herbicide Formulations 

(Ester Forms of Hormone-Type) 
Volatility Determination 

First Action 1960 
Final Action 1961 

A. Material 

(a) Paper bags. — No. 20 to open with flat bottom. Close 
with paper clips. 

(b) Filter paper. — 7 cm diam. 

(c) Bacteriological loop. — 0.01 mL. Wash with acetone af- 
ter each application or heat to cherry red in flame. 

(d) Test plants. — Actively growing tomato seedlings 65— 
75 mm high in 3-4" pots. 

(e) Formulation to be tested. — Use 0.01 mL aliquot of 4 
lb/gal. formulation or equiv. vol. of other concns. 

(f) High and low volatile ester stds. — Use Bu ester of 2,4-D 
as high volatile ester and tetrahydrofurfural ester of 2,4-D as 
low volatile ester with same wt of acid /gal. as formulations 
to be tested. 



B. Operating Technic 

Open bags with flat bottom and place plant toward one side 
on bottom of bag. Apply 0.01 mL of formulation to middle 
of filter paper by means of bacteriological loop, and for con- 
trols, apply 0.01 mL solv. only. Place treated paper in bottom 
of bag. Do not touch treated part of paper against plant, sides 
of bag, or pot. Close bag by folding top, secure with clips, 
and let stand 24 hr at 85-1 10°F (29-43°C). 

Use 3 plants per treatment and 3 for controls. Repeat test 
on another day. 

Remove plants from bag, let stand 24 hr, and read curvature 
(stem bending, epinasty) response. (Fold and discard used bags 
to prevent contamination.) Rate plants according to scale as 
follows: 

(1) Normal growth of untreated check- no apparent re- 
sponse. 

(2) Epinasty 1-20° compared to normal- no curling. 

(3) Epinasty 21-40° compared to normal-slight curling. 

(4) Epinasty 41-60° compared to normal-moderate curling. 

(5) Epinasty 61-80° compared to normal-moderate curling. 

(6) Epinasty 81 to >90° compared to normal-severe form- 
ative effects. 

Mean response of 1 to 2.4 for all tests indicates low vola- 
tility. Mean response of 2.5 to 6 indicates volatile formulation. 

To detect small differences between low volatile esters, or 
differences between 2,4-D and 2,4,5-T types, hold plants 7 
days after treatment to allow time for modified leaves or stem 
lesions to develop. Absence of such responses indicates that 
formulation was a low volatile 2,4,5-T ester. 

Ref.: J AOAC 43, 367(1960). 



INORGANIC AND ORGANOMETALL1C 
PESTICIDES AND ADJUVANTS 



920.13 



Paris Green Pesticide 

Formulations 

Final Action 



A. Moisture 
See 920.12. 

B. Totai Arsenic 
See 922.03. 

C. Water-Soluble Arsenious Oxide* 
—Surplus 1965 

See 4.031, 10th ed. 



920.14* Paris Green Pesticide 

Formulations 
Total Arsenious Oxide 

Final Action 
Surplus 1965 

(Following methods det. only As present in trivalent form 

(As 2 3 ) and Sb present in trivalent form (Sb 2 3 ) in absence 

of ferrous and cuprous salts.) 

A. Method I 

See 4.028-4.029, 10th ed. 

B. Method II 

See 4.030, 10th ed. 



154 



Pesticide Formulations 



AOAC Official Methods of Analysis (1990) 



920.15* Paris Green Pesticide 

Formulations 
Total Copper 

Final Action 
Surplus 1965 

A. Electrolytic Method 
See 4.032, 10th ed. 

B. Volumetric Thiosulfate Method 

See 4.033, 10th ed. 



920.16 Lead Arsenate 

Pesticide Formulations 

Moisture 

Final Action 

{Caution: See safety notes on pesticides and arsenic trioxide.) 

(a) Powder. — Dry 2 g to const wt at 105-110°. Report loss 
in wt as H 2 0. 

(b) Paste. — Proceed as in (a), using 50 g. Grind dry sam- 
ple to fine powder, mix well, transfer small portion to sample 
bottle, and again dry 1-2 hr at 105-110°. Use this anhyd. 
material to det. total Pb and total As. 



920.17 Lead Arsenate 

Pesticide Formulations 
Total Arsenic 
Final Action 

A. Method I 
See 922.03C. 

B. Method II 

(Not applicable in presence of Sb) 

Dissolve 1 g powd sample with HN0 3 (1+4) in porcelain 
casserole or evapg dish, add 5 mL H 2 S0 4 , and heat on hot 
plate to copious evolution of white fumes. Cool, add little H 2 0, 
and again evap. until white fumes appear, to assure removal 
of last trace of HN0 3 . Wash into 200 mL vol. flask with H 2 0, 
cool, dil. to vol., and filter thru dry filter. Transfer 100 mL 
filtrate to erJenmeyer and proceed as in 925.02, beginning w . . . 
add 1 g KI, ..." From mL std 1 soln used, calc. % total As 
as As 2 5 . 

Ref.: USDA Bur. Chem. Bull. 105, p. 167. 

CAS-7440-38-2 (arsenic) 



920.18 Lead Arsenate 

Pesticide Formulations 

Total Arsenious Oxide 

Final Action 

Weigh 2 g powd sample and transfer to 200 mL vol. flask, 
add 100 mL H 2 S0 4 (1 + 6), and boil 30 min. Cool, dil. to vol., 
shake thoroly, and filter thru dry filter. Nearly neutze 100 mL 
filtrate with NaOH soln, 922.03A(e), using few drops phthln. 
If neut. point is passed, make acid again with the dil. H 2 S0 4 . 
Continue as in 925.02, beginning "Neutze with NaHC0 3 , ..." 
From mL std I soln used, calc. % As 2 3 . 

Ref.: JAOAC 3, 332(1920). 



920.19 Lead Arsenate 

Pesticide Formulations 

Total Arsenic Oxide 

Final Action 

A. Reagents 

(a) Potassium iodide soln. — Dissolve 20 g Kl in H 2 and 
dil. to 100 mL. 

(b) Thiosulfate std soln.— 0.05N. Prep, daily by dilg O.liV 
soln, 942.27. 1. mL 0.057V Na 2 S 2 3 = 2.873 mg As 2 O s . 

B. Determination 

Weigh 0.5 g powd sample and transfer to erlenmeyer. Add 
25-30 mL HC1 and evap. to dryness on steam bath. Add 50 
mL HO and if necessary to effect soln, heat on steam bath, 
keeping flask covered with watch glass to prevent evapn of 
acid. Cool to 20-25°, add 10 mL of the KI soln and 50 mL 
(or more if necessary to produce clear soln) 25% NH 4 Cl soln, 
and immediately titr. liberated I with std Na 2 S 2 3 soln. When 
color becomes faint yellow, dil. with ca 150 mL H 2 and con- 
tinue titrn carefully, dropwise, until colorless, using starch in- 
dicator, 922.03A(f), near end point. From mL Na 2 S 2 3 soln 
used, calc. % As 2 5 . 

Ref.: JAOAC 3, 333(1920). 



920.20 Lead Arsenate 

Pesticide Formulations 

Water-Soluble Arsenic 

Final Action 

Proceed as in 925.02, and calc. results as As 2 5 . 



920.21 Lead Arsenate 

Pesticide Formulations 
Total Lead 
Final Action 

In 600 mL beaker on hot plate heat 0.5 g powd sample and 
ca 25 mL HN0 3 (1+4). Filter to remove any insol. residue. 
Dil. to >400 mL, heat nearly to bp, and add NH 4 OH to slight 
pptn, then HN0 3 (1 + 9) to redissolve ppt, adding 1-2 mL ex- 
cess. Into this soln, kept almost boiling, pipet 50 mL hot 10% 
K 2 Cr0 4 soln, stirring constantly. Decant while hot thru weighed 
gooch, previously heated to 140-150°, and wash ppt several 
times by decanting and then on filter with boiling H 2 until 
washings are colorless. Dry PbCr0 4 at 140-150° to const wt. 
From wt PbCr0 4 , calc. % Pb, using factor 0.641 1. (PbCr0 4 
ppt may contain small amt PbHAs0 4 , which will cause slightly 
high results, but this error rarely is >0. 1.-0.2%.) 

Refs.: USDA Bur. Chem. Bull. 137, p. 40; 152, p. 68. 

CAS-7439-92-1 (lead) 



921.05 



Calcium Arsenate 

Pesticide Formulations 

Final Action 



{Caution: See safety notes on pesticides.) 

A. Moisture 
See 920.12. 

B. Total Arsenic 
See 922.03. 



AOAC Official Methods of Analysis (1990) 



Inorganics and Organometallics 1 55 



C. Total Arsenious Oxide 

(a) Not applicable in presence of nitrates. — Weigh 1 g 
sample, transfer to 500 mL erlenmeyer, and dissolve in 100 
mL HC1 (.1+3). Heat to 90° and titr. with std KBr0 3 soln, 
922.03A(c), using 10 drops Me orange, 922.03A(g). From mL 
std KBr0 3 soln used, calc. % As 2 3 . 

(b) Applicable in presence of small amounts of nitrates. — 
Proceed as in (a) except to titr. at room temp. 

Refs.: JAOAC 5, 33(1921); 6, 392(1922). 

D. Water-Soluble Arsenic 

Proceed as in 925.02, and calc. results as As 2 5 . (In testing 
Ca arsenate by this method, low value for H 2 0-sol. As is not 
assurance against plant injury when using this product.) 



921.06 Calcium Arsenate 

Pesticides Formulations 

Total Calcium 

Final Action 

A. Reagents 

(a) Ammonium oxalate soln. — Dissolve 40 g (NH^CoCV 
H 2 in 1 L H 2 0. 

(b) Potassium permanganate std soln. — 0.17V. Prep, and 
stdze as in 940.35. 

R Method l 

Dissolve 2 g sample in 80 mL HOAc (1 +3), transfer to 200 
mL vol. flask, dil. to vol., and filter thru dry filter. Transfer 
50 mL aliquot to beaker, dil. to ca 200 mL, heat to bp, and 
ppt Ca with (NH 4 ) 2 C 2 4 soln. Let beaker stand 3 fir on steam 
bath, filter, and wash ppt with hot H 2 0. Dissolve ppt in 200 
mL H 2 contg 25 mL H 2 S0 4 (1 +4), heat to ca 70°, and titr. 
with std KMn0 4 soln. From mL KMn0 4 soln used, calc. % 
Ca. 

C. Method It 

(Not applicable in presence of Pb. Caution: See safety notes 
on arsenic trioxide.) 

Weigh 2 g sample, transfer to beaker, add 5 mL HBr (ca 
1.38 sp gr) and 15 mL HO, and evap. to dryness under hood 
to remove As. Repeat treatment, add 20 mL HC1, and again 
evap. to dryness. Take up with H 2 and little HO, filter into 
200 mL vol. flask, wash, and dil. to vol. Transfer 50 mL ali- 
quot to beaker, add 10 mL HO and few drops HN0 3 , boil, 
and make slightly alk. with NH 4 OH. Let stand few min and 
filter. Dissolve ppt in HO (1 +4), reppt, filter thru same paper, 
and wash with hot H 2 0. To combined filtrates and washings 
add 20 mL HOAc (1 + 3) and adjust to ca 200 mL. Heat to bp, 
ppt with (NH 4 ) 2 C 2 4 soln, and let stand 3 hr on steam bath. 
Filter, and wash with hot H 2 0. Ignite at 950°, and weigh as 
CaO; or dissolve and titr. as in 921. 06B. From wt CaO or mL 
KMn0 4 soln used, calc. % Ca. 

Refs.: JAOAC 5, 33(1921); 6, 392(1922). 

CAS-7440-70-2 (calcium) 



920.22 



A. Moisture 
See 920.12. 



Zinc Arsenite 
Pesticide Formulations 

Final Action 



B. Total Arsenic 

Proceed as in 922.03 and calc. as As 2 3 . 

C. Water-Soluble Arsenic 

Proceed as in 925.02, and calc. results as As 2 3 . 

D. Total Zinc* 
Surplus 1965 

See 4.054, 10th ed. 



920.23* Zinc Arsenite 

Pesticide Formulations 
Total Arsenious Oxide 

Final Action 
Surplus 1965 

A. Method I 

See 4.051, 10th ed. 

S. Method tl 
See 4.052, 10th ed. 



920.24* Copper in Copper Carbonate 

Pesticide Formulations 

Final Action 
Surplus 1965 

A. Electrolytic Method 
See 4.055, 10th ed. 

B. Volumetric Thiosulfate Method 
5<?<? 4.056, 10th ed. 



964.03 Copper in Copper Nap ht hen ate 

Pesticide Formulations 

First Action 1964 
Final Action 1982 

(Caution: See safety notes on pesticides.) 

A. Titrimetric Method 

Accurately weigh sample contg ca 0.2 g Cu into dry g-s 
flask. Add 5 mL pet ether to coned products. Add 100 mL 
H 2 0, 1.5 g NH 4 HF 2 , and 5-10 g KI. Stopper and shake vig- 
orously until reaction is complete (usually ca 2 min). Wash 
stopper and sides of flask with H 2 and titr. with std O.IjV 
Na 2 S 2 3 (stdzd against Cu) to light brown. Add starch indi- 
cator, 922.03A(f), titr. almost to end point, add 2 g KSCN, 
shake to dissolve, and complete titrn to starch end point. 

a Electrolytic Method 

Accurately weigh sample contg ca 0.2 g Cu into 200 mL 
separator. Add 50 mL pet ether and 25 mL HN0 3 (1+4), and 
shake 2 min. Drain aq. phase into 250 mL beaker and save. 
Wash pet ether with 15 and 10 mL HN0 3 (1+4), and combine 
acid exts. Neutze with NH 4 OH, acidify with 6 mL H 2 S0 4 and 
4 mL HN0 3 , and proceed as in 922. 05 A, beginning ". . . ad- 
just vol. to 200 mL, ..." using ca 0.5 amp during first 10 
min and 1 .5-2.0 amp for ca 20 min. 

Ref.: JAOAC 47, 253(1964). 

CAS-7440-50-8 (copper) 



156 



Pesticide Formulations 



AOAC Official Methods of Analysis (1990) 



981.01 Copper (Water-Soluble) 

in Water-insoluble Copper Fungicides 

First Action 1981 

CIPAC-AOAC Method 

(Caution: See safety notes on atomic absorption spectropho- 
tometer and chloroform.) 

A. Principie 

Water-soluble Cu is isolated by dispersing sample in H 2 0, 
shaking, centrifg, and filtering. Cu in filtrate is detd either by 
spectrophtric bathocuproine method or by atomic absorption. 

B. Apparatus 

(a) Filter paper. — 7 cm. (Whatman No. 1 chromatgc or 
filter paper, S&S No. 2043b, and Albet No. 305 have been 
satisfactory; the following papers have not been suitable: 
Whatman Nos 32, 40, 42, and Albet No. 240. If other papers 
are used, check adsorption of Cu before use.) 

(b) Filter.— 13 mm Millipore, HA.WP.013-porosity 0.45 
|xm, Sartorius, or equiv. 

(c) Atomic absorption spectrophotometer. — With oxidizing 
air-C 2 H 2 flame. 

C. Reagents 

(a) Deionized water. — For prepg all aq. solns and rinsing 
glassware and app. 

(b) Sodium acetate-acetic acid. — 0.05M, buffered at pH 6.5. 
Dissolve 3.4 g NaOAc.3H 2 in H 2 0, add 1.8 mL 0.2M HOAc, 
adjust to pH 6.5, and dil. to 500 mL with H 2 0. 

(c) Cupric sulfate.— CuS0 4 .5H 2 0, contg 25.4% Cu. Check 
Cu content, e.g., by electrolysis. 

(d) Copper std stock so In. — 25 |mg Cu/mL. Dissolve 98.3 
mg CuS0 4 .5H 2 in H 2 and dil. to 1 L with H 2 0. 

(e) Ascorbic acid soln. — Dissolve 10 g ascorbic acid in H 2 
and dil. to 100 mL with H 2 0. 

(f) Sodium acetate soln. — Dissolve 40 g NaOAc.3H 2 in 
H 2 and dil. to 100 mL with H 2 0. 

(g) Chloroform. — Redistd. 

(h) Bathocuproine soln. — Dissolve 0.050 g in 500 mL re- 
distd CHC1 3 and store in dark glass bottles. 

D. Isolation of Water-Soiuble Copper 

Accurately weigh ca 0.350 g sample (w) and transfer to 250 
mL conical flask. Add 100 mL H?0, stopper tightly, and shake 
1 min. Let stand l.h in 20° H 2 bath, shaking suspension 
every 15 min. Centrf. ca 50 mL suspension 20 min at 3000 
rpm, taking care to avoid increase in temp. Filter ca 40 mL 
supernate thru 13 mm filter. 



Bathocuproine Method 

E. Preparation of Standard Curve 

Pipet 25.0 mL Cu std stock soln, (d), into 500 mL vol. flask 
and dil. to vol. with NaOAc-HOAc buffer soln (1.25 (Jig Cu/ 
mL). Transfer 0, 4, 6, 8, 10, 12, 14, and 16 mL aliquots of 
intermediate soln to 100 mL separators and dil. each to 20 mL 
with NaOAc-HOAc buffer soln. Add 1 mL ascorbic acid soln 
to each separator and shake 1 min. Add 2.5 mL NaOAc soln 
and shake 1 min. Develop color by pipetting in 10.0 mL 
bathocuproine soln. Ext. complex by vigorously shaking 1 min 
(vigorous extn is necessary to ext all complex). Let layers sep., 
filter CHCI3 ext thru 7 cm paper into clean, dry tube, and stop- 
per tube. Exactly 15 min after extn, measure A at 465 nm, 
using CHCI3 as ref. Plot A vs jxg Cu. (Curve is linear <20 |xg 
Cu, but does not necessarily pass thru origin.) 



F. Determination 

Pipet 10.0 mL sample filtrate into 100 mL vol. flask, dil. 
to vol. with NaOAc-HOAc buffer soln, and mix. 

Transfer 20.0 mL aliquot to 100 mL separator and continue 
as in Preparation of Standard Curve , starting with "Add 1 mL 
ascorbic acid soln ..." 

If A of sample is higher than A of highest point of calibration 
curve, diL sample soln with buffer soln so A falls on calibra- 
tion curve. Read u,g Cu (0 corresponding to A found. 

% Soluble Cu (w/w) - 2/200w 

If soln has been dild, correct formula accordingly. Carry out 
blank detn on reagents, using 20 mL NaOAc-HOAc buffer 
soln. A must be of same order as that for A of point on 
calibration curve. 



Atomic Absorption Spectrophotometry Method 

G. Preparation of Standard Curve 

Transfer 0, 4.0, 8.0, 12.0, 16.0, and 20.0 mL aliquots of 
Cu std stock soln (25 u>g Cu/mL) to 100 mL vol. flasks. Add 
2 mL HNO3 to each, diL to vol. with H 2 0, and mix. (These 
solns contain 0, 1, 2, 3, 4, and 5 u,g Cu/mL.) 

Det. A at 324.7 nm of solns in oxidizing air-C 2 H 2 flame, 
and plot calibration curve. 

H. Determination 

Pipet 25.0 mL sample filtrate into 50 mL vol. flask, add 1 
mL HNO3, dil. to vol. with H 2 0, and mix. Det. A at 324.7 
nm as in Preparation of Standard Curve. Obtain fig Cu/mL 
from std curve, 

% Soluble Cu (w/w) - (pig Cu/mL)/50w 

If A of sample is higher than highest point of calibration curve, 
dil. sample with 2% HNO3 and correct calcn for diln factor. 

Ref.: JAOAC 64, 75(1981); corr. 233. 



CAS-7440-50-8 (copper) 



979.02 Fentin in Pesticide Formulations 

Potentiometric Titration Method 

First Action 1979 
Final Action 1982 

CIPAC-AOAC Method 

A. Principle 

Org. Sn compds are extd with acetone, diphenyltin compds 
are quant, converted to insol. oxide with alk. alumina and fil- 
tered, and acetone soln is titrd potentiometric ally. 

B. Apparatus 

(a) Filtration apparatus. — Glass bell with neck and remov- 
able plate to permit glass buchner with fine porosity fritted 
disk and long stem to drain into beaker under vac. (Fig. 979.02). 

(b) Potentiometric titration apparatus. — pH meter with glass 
and satd calomel electrodes is satisfactory. 

C. Reagents 

(a) Alkaline alumina. — Mix 150 g neutral A1 2 3 (Woeim 
4649, or equiv.) with 150 mL alcohol contg 15 g KOH in 1 
L r-b flask. Reflux 30 min, cool, and filter with suction thru 
buchner. Dry powder in vac. 1 hr at 100° and 3-3.5 hr at 130°. 
Pour warm powder into bottle and stopper tightly. Com. alk. 
A1 2 3 is not satisfactory. 

(b) Cellulose powder. — Whatman CF 11, or equiv. 



AOAC Official Methods of Analysis (1990) 



Inorganics and Organometallics 



157 




FIG. 979.02— Filtration apparatus 



D. Determination 

Accurately weigh into 100 mL glass beaker sample contg 
ca 0.30 g triphenyltin compd. Add 2 g alk. A1 2 3 and 25 mL 
acetone and stir with mag. stirrer 10 min. Prep, and process 
blank of 2 g alk. A1 2 3 and 25 mL acetone in same manner. 
Place 1 g cellulose powder and 1 g alk. A1 2 3 in funnel and 
mix thoroly. Assemble filtration app. contg 250 mL beaker 
and filter suspension thru funnel. Wash beaker and funnel with 
four 20 mL portions acetone. Titr. filtrate potentiometrically 
with stdzdO.UVHCl. 

% Triphenyltin compd = (S - B) X N X (M/W) X 10 

where S = mL HC1 used for sample, B = mL HC1 used for 
blank, N = normality of HC1, M = mol. wt of compd (367.0 
for triphenyltin hydroxide and 409.0 for the acetate), and W 
— g sample. 

Ref.: JAOAC61, 1504(1978). 



984.04 Fentin in Fentin-Maneb 

Pesticide Formulations 

Gas Chromatographic Method 

First Action 1984 

CIPAC-AOAC Method 

A. Principie 

Fentin is converted with n-butylmagnesium chloride to bu- 
tyltriphenyltin, which is extd with toluene and detd by GC 
with docosane as internal std. 

B. Reagents and Apparatus 

(a) Dilute acetic acid. — Weigh 0.750 g HO Ac in 250 mL 
vol. flask and dil. to vol. with toluene. 

(b) n-Butylmagnesium chloride. — 2.5M in tetrahydrofuran 
(Alfa Products, Div. of Morton Thiokol, Inc., 152 Andover 
St, Danvers, MA 01923), or equiv. 

(c) Butyltriphenyltin. — Prep, as follows: Purify triphenyltin 
chloride by repeated crystn from isopropanol until diphenyltin 
dichloride and/or tetraphenyltin are absent by GC or TLC. 
(For TLC, use silica gel F254 (Merck) plates and develop in 
CH 2 Cl 2 -acetone-HOAc-H 2 (80 + 5 + 2.5 + 0.5) or CH 2 C1 2 - 
n-hexane-HOAc (30 + 20 + 2.5). Expose developed plates to 
UV radiation (254 nm) or I vapors and spray with 0.1% pyro- 
catechol violet soln in alcohol.) Dry salt at 50° under vac. to 
remove isopropanol. Flush 250 mL 3 -necked flask equipped 



with stirrer, reflux condenser, and dropping funnel with N. 
Dissolve 20 g triphenyltin chloride in 80 mL toluene, add 40 
mL n-butylmagnesium chloride, and let react 15 min under N, 
Carefully destroy excess Grignard reagent (cool in H 2 bath) 
by adding 50 mL 5% H 2 S0 4 and transfer mixt to sep. funnel. 
Discard lower (aq.) layer. Wash org. layer with 20 mL 5% 
H 2 S0 4 and then with H 2 until acid-free. Dry org. layer with 
anhyd. Na 2 S0 4 . Filter soln into 500 mL r-b flask and evap. 
solv. in rotary vac. evaporator at 50°. Dissolve residue in 400 
mL hot isopropanol, cool, and filter butyltriphenyltin in buch- 
ner funnel; wash with 25 mL cold isopropanol and dry at 50° 
under vac. Check purity by GC and mp (corrected mp 60.0- 
60.5°). 

(d) Internal std soln. — Accurately weigh 2.25 g pure do- 
cosane into 250 mL vol. flask, and dissolve and dil. to vol. 
with toluene. 

(e) Calibration soln. — Accurately weigh ca 0.20 g pure bu- 
tyltriphenyltin into g-s flask, pipet in 10.0 mL internal std soln, 
and add 40 mL toluene. 

(f ) Gas chromato graph. — With on-column injection, flame 
ionization detector, and 1.5 m X 3 mm (id) glass column packed 
with 5% SE-30, or equiv. on 100-200 mesh Chromosorb W 
(HP) or Gas-Chrom Q. Operating conditions: temps (°): col- 
umn 220, injection port 250, detector 260; flow rates (mL/ 
min): N carrier gas 30, air 400, H 40. Alternatively, thermal 
conductivity detector may be used with He carrier gas, bridge 
current at 150 mamp, and detector at 300°. 

C. Preparation of Sample 

(a) Fentin acetate -rnaneb mixtures. — Accurately weigh 
sample contg 205 ± 5 mg fentin acetate into 100 mL erlen- 
meyer. Pipet in 10.0 mL internal std soln and 15 mL toluene 
and stir 5 min on mag. stirrer. Add 8 mL n-butylmagnesium 
chloride and let react 15 min under N. Destroy excess Grig- 
nard reagent by careful and dropwise addn of 1.5 mL H 2 by 
syringe or microburet. Dry dispersion with 2 g anhyd. Na 2 S0 4 . 
Add 25 mL toluene (omit if thermal conductivity detector is 
used) and filter. 

(b) Fentin hydroxide -maneb mixtures. — Accurately weigh 
sample contg 185 ± 5 mg fentin hydroxide into 100 mL er- 
lenmeyer. Pipet in 10.0 mL internal std soln and 1.0 mL dil. 
HO Ac. Add 5 mL toluene and stir 5 min on mag stirrer. Con- 
tinue as described in (a). 

D. Determination 

Inject 1 |xL portions of calibration soln until peak area ratio 
(area butyltriphenyltin peak/area docosane peak) varies <1% 
for successive injections. Inject, in duplicate, 1 |xL portions 
of sample solution followed by 1 jxL portions of calibration 
soln. (For thermal conductivity detection, inject 10-20 |xL.) 
Measure peak areas by tri angulation (peak ht by retention time) 
or by electronic integration. Retention times: internal std 7 min; 
butyltriphenyltin 14 min. 

E. Calculations 

Fentin acetate, % = R/R' x W /W x 100.5 
Fentin hydroxide, % - R/R' x W'/W x 90.2 

where R and R' — av. peak area ratios for sample and cali- 
bration solns, resp.; W and W — mg sample and butyl- 
triphenyltin, resp.; 100.5 = 100 x mol. wt fentin acetate/ 
mol. wt butyltriphenyltin = 100 X 409/407; 90.2 - 100 x 
mol. wt fentin hydroxide /mol. wt butyltriphenyltin = 100 X 
367/407. 

Refs.: JAOAC 61, 1507(1978); 67, 479(1984). 

CAS-900-95-8 (fentin acetate) 
CAS-76-87-9 (fentin hydroxide) 



158 



Pesticide Formulations 



AOAC Official Methods of Analysis (1990) 



920.25 Moisture in Bordeaux Mixture 

Pesticide Formulations 
Final Action 

(Caution: See safety notes on pesticides.) 

(a) Powder.— See 920.16(a). 

(b) Paste.— Heat ca 100 g in oven at 90-100° until dry 
enough to powder readily and note loss in wt. Powder this 
partially dried sample and det. remaining H 2 in 2 g as in (a). 
Det. C0 2 as in 920. 26B, both in original paste and in partially 
dried sample. Calc. total H 2 by following formula: 

, (100-fl)(6 + c) _ 

M = a + d 

100 

where M = % total H 2 in original paste; a = % loss in wt 
of original paste during first drying; b = % loss in wt of par- 
tially dried paste during second drying; c — % C0 2 remaining 
in partially dried paste after first drying; and d = % total C0 2 
in original paste. 



920.26 Carbon Dioxide 

in Bordeaux Mixture 

Pesticide Formulations 

Final Action 

A. Apparatus 

Use 200 ml erlenmeyer with 2-hole stopper; in one hole fit 
dropping funnel with stem extending almost to bottom of flask, 
and thru other hole pass outlet of condenser that is inclined 
upward at 30° angle from horizontal. Connect upper end of 
condenser with CaCl 2 tube, which in turn connects with double 
U-tube filled in middle with pumice fragments, previously satd 
with 20% CuS0 4 .5H 2 soln and subsequently dehydrated, and 
with CaCl 2 at either end. Connect 2 weighed U-tubes to absorb 
C0 2 , first filled with porous soda-lime, and second, l /a with 
soda-lime and 2 / 3 with CaC1 2 , placing the CaCl 2 at exit end of 
train. Attach Geissler bulb, partly filled with H 2 S0 4 , to last 
U-tube to show rate of gas flow, and connect aspirator with 
Geissler bulb to draw air thru app. Connect absorption tower 
filled with soda-lime to mouth of dropping funnel to remove 
C0 2 from air entering app. 

B. Determination 

Weigh 2 g powder or 10 g paste into the erlenmeyer and 
add ca 20 mL H 2 0. Attach flask to app. , omitting the 2 weighed 
U-tubes, and draw C0 2 -free air thru app. until it displaces 
original air. Attach weighed U-tubes as in 920. 26A, close 
stopcock of dropping funnel, pour into it 50 mL HC1 (1+4), 
reconnect with soda-lime tower, and let acid flow into erlen- 
meyer, slowly if there is much C0 2 , rapidly if there is little. 
When effervescence diminishes, place low Bunsen flame un- 
der flask and start flow of H 2 thru condenser, letting slow 
current of air flow thru app. at same time. Maintain steady but 
quiet boil and slow air current thru app. Boil few min after 
H 2 begins to condense, remove flame, and continue air flow 
at ca 2 bubbles/sec until app. is cool. Disconnect weighed 
absorption tubes, cool in balance case, and weigh. Increase in 
wt = C0 2 . 



920.27 Copper in Bordeaux Mixture 

Pesticide Formulations 



Refs.: Fresenius, "Quantitative Chemical Analysis,' 
6th German Ed., 1906, amplified and revised, 
1180. U.S. Geol. Survey Bull. 700, p. 218. 

CAS- 124-38-9 (carbon dioxide) 



Trans. 
Vol. 2, 



Final Action 



A. Electrolytic Method 



(Also applicable to CuC0 3 and CuS0 4 ) 

Dissolve powd sample contg 0.2-0.25 g Cu in 45 mL HNO3 
(1+4). Filter if necessary, dil. to 200 mL, and electrolyze as 
in 922.05A. 

B. Volumetric Thiosulfate Method 

Dissolve 2 g powd sample in ca 25 mL H.NO3 (1+4), dil. 
to 50 mL, add NH 4 OH in excess, and heat. Without removing 
ppt that has formed, boil off excess NH 3 , add 3-4 mL HO Ac, 
cool, add 10 mL 30% Kl soln, and titr. as in 922.05B, be- 
ginning "... titr. with std Na 2 S 2 3 soln, ..." 

CAS-7440-50-8 (copper) 



920.28 



Bordeaux Mixture 

with Paris Green 

Final Action 



(Caution: See safety notes on pesticides.) 

A. Moisture 
See 920.25. 

B. Carbon Dioxide 
See 920.26B. 

C. Total Arsenic 

Proceed as in 922.03, using 2 g sample, and calc. results 
as As 2 3 . 

D. Total Arsenious Oxide* 
—Surplus 1965 

See 4.067, 1 0th ed. 

E. Water-Soluble Arsenious Oxide* 
Surplus 1965 

See 4.068, 10th ed. 



Copper 



F. Electrolytic Method I 
See 922.05A. 



G. Electrolytic Method II— (Short Method)* 
—Surplus 1965 

See 4.070, 10th ed. 

H. Volumetric Thiosulfate Method 
See 922.05B. 



920.29 



Bordeaux Mixture 

with Lead Arsenate 

Final Action 



(Caution: See safety notes on pesticides.) 

A. Moisture 
See 920.25. 

B. Carbon Dioxide 
See 920.26B. 



AOAC Official Methods of Analysis (1990) 



Inorganics and Organometallics 1 59 



C. Total Arsenic 

Proceed as in 922.03, using 2 g sample, and calc. results 
as As 2 5 . 

D. Water-Soluble Arsenic 

Proceed as in 925.02 and calc. results as As 2 5 . 

E. Copper— Electrolytic Method 
See 922.05A 

F. Copper— Volumetric Thiosulfate Method 
See 922.05B. 

G. Lead 

See 922.04. 

H. Lead and Copper — Electrolytic Method* 
—Surplus 1965 

See 4.079-4.080, 10th ed. 



925.03 



Bordeaux Mixture 

with Calcium Arsenate 

Final Action 



952.01 Potassium Cyanate 

Pesticide Formulations 
Final Action 

(Caution: See safety notes on pesticides.) 

A, Reagent 

Wash soln. — Satd aq. soln of hydrazodicarbamide, 
NH 2 CONHNHCONH 2 . Prep, by mixing KOCN and semicar- 
bazide.HCl, NH 2 CONHNH 2 .HCl, in H 2 0, filter, and wash ppt 
with H 2 0. Transfer ppt to flask, add small amt H 2 0, shake 
vigorously, and filter. (Solubility of ppt in H 2 is ca 1 part in 
6600.) 

B. Determination 

Weigh sample contg 0.2-0.5 g KOCN into 100 mL beaker, 
add 20 mL wash soln and 1 g semicarbazide.HCl, and let stand 
24 hr. Filter hydrazodicarbamide on gooch or fine fritted glass 
crucible, wash with 10 mL wash soln, and dry at 100° to const 
wt. KOCN = wt residue x 0.6868, 

Ref.: JAOAC 35, 377(1952). 

CAS-590-28-3 (potassium cyanate) 



(Caution: See safety notes on pesticides.) 

A. Moisture 
See 920.25. 

B. Carbon Dioxide 
See 920.26B 

C. Total Arsenic 

Proceed as in 922.03, using 2 g sample, and calc. results 
as As 2 O s . 

D. Water-Soluble Arsenic 

Proceed as in 925.02 and calc. results as As 2 5 . 

Copper 

E. Electrolytic Method I 
See 922.05A. 

F. Electrolytic Method //* 
—Surplus 1965 

See 4.070, 10th ed. 

G. Volumetric Thiosulfate Method 
See 922.05B. 



930. 12* Calcium Cyanide 

Pesticide Formulations 



Final Action 
Surplus 1965 



A. Cyanide 

See 4.093-4.094, 10th ed. 



Chloride 



B. Method I 

See 4.095, 10th ed. 

C. Method II 

See 4.096, 10th ed. 



920.30* Sodium and Potassium Cyanide 
Pesticide Formulations 

Final Action 
Surplus 1965 

A. Cyanide 

See 4.088-4.089, 10th ed. 

Chloride 

B. Method I 

See 4.090-4.091, 10th ed. 

C. Method II 

See 4.092, 10th ed. 



920.31 Sulfur (Soluble) in Lime Sulfur 

Solutions and Dry Lime Sulfur 
Gravimetric Method 
Final Action 

(Use low S reagents.) 

A. Preparation of Sample 

(a) Solns. — Accurately weigh ca 10 g soln, transfer to 250 
mL vol. flask, and immediately dil. to vol. with recently boiled 
and cooled H 2 0. Mix thoroly and either take necessary ali- 
quots in individual pipets in mi n. time for detns or transfer to 
small bottles, filling them completely and avoiding contact of 
soln with air as much as possible. Stopper bottles, seal with 
paraffin, and store in dark, cool place. 

(b) Dry lime-sulfur. — Thoroly stir 5 g sample with ca 50 
mL H 2 in 250 mL beaker. Let settle and decant thru paper 
into 250 mL vol. flask. Repeat extn with H 2 until filtrate is 
colorless and ca 200 mL is obtained. Transfer residue to paper, 
wash with hot H 2 0, cool to room temp., and dil, to vol. Dry 
residue 1.5 hr at 105°, and reserve for free S and sulfite S 
detns in residue, if desired. (Ext S from dry residue with CS 2 
(Caution: See safety notes on flammable solvents, toxic sol- 
vents, and carbon disulfide.), evap. on steam bath or in air 
current, dry 15 min at 105°, weigh, and calc. % S.) 



160 



Pesticide Formulations 



AOAC Official Methods of Analysis (1990) 



Prep, so In in min. time and keep beaker and funnel covered 
as much as possible. 

B. Determination 

With clean, dry pipet transfer 10 mL prepd soln, 920.31A(a) 
or (b), to 250 mL beaker. Partially cover with cover glass and 
add 2-3 g Na 2 2 in small portions, with stirring, from tip of 
spatula. Continue adding Na 2 2 until all S appears to be ox- 
idized to sulfate (yellow color disappears). Add slight excess 
Na 2 2 , completely cover beaker with cover glass, and heat on 
steam bath, stirring occasionally, 15-20 min. 

Wash off cover glass and sides of beaker, acidify with HC1 
(1+4), and filter if necessary. Dil. to 150-200 mL, heat to 
bp, and add 10% BaCl 2 soln (11 mL/1 g BaS0 4 ), with const 
stirring, at such rate that ca 4 min is required to add necessary 
amt. Let stand until clear and cool, filter thru quant, paper, 
wash until CI- free, ignite carefully, and heat to const wt over 
Bunsen burner. Calc. % S from wt BaS0 4 , using factor 0.1374. 

Ref.: JAOAC 3, 353(1920). 

CAS-7704-34-9 (sulfur) 



920.32 Sulfur (ThiosuSfate) 

in Lime Sulfur Solutions 

and Dry Lime Sulfur 

Titrimetric Method 

Final Action 

A. Reagent 

Ammoniacal zinc chloride soln. — Dissolve 50 g ZnCl 2 in ca 
500 mL H 2 0, add 125 mL NH 4 OH and 50 g NH 4 C1, and dil. 
to 1 L. 

B. Determination 

To 50 mL H 2 in 200 mL vol. flask add 50 mL prepd soln, 
920.31A(a) or (b). Add slight excess of the ammoniacal ZnCl 2 
soln and dil. to vol. Complete detn as rapidly as possible. Shake 
thoroly and filter thru dry filter. To 100 mL filtrate add few 
drops Me orange, 922.03A(g), or Me red (1 g Me red in 200 
mL alcohol), and exactly neutze with O.iyV HC1. Titr. neut. 
soln with 0.057V 1, 922.03A(b), using few drops starch indi- 
cator, 922. 03 A (f). From mL 1 soln used, calc. % thiosulfate 
S present. (Factor of I soln in terms of As 2 3 x 1.296 = equiv. 
in thiosulfate S.) 



B. Indirect Method 

Difference between sol. S and sum of thiosulfate S and sul- 
fate S - sulfide S. 



Ref.: JAOAC 3, 353(1920). 



920.33 Sulfur (Sulfide) 

in Lime Sulfur Solutions 

and Dry Lime Sulfur 

Final Action 

A. Zinc Chloride Method 

To 10-15 mL H 2 in small beaker add 10 mL aliquot prepd 
soln, 920.31A(a) or (b). Calc. amt ammoniacal ZnCl 2 soln, 
920. 32A, necessary to ppt all S in aliquot and add slight ex- 
cess. Stir thoroly, filter, wash ppt twice with cold H 2 0, and 
transfer paper and ppt to beaker in which pptn was made. Cover 
with H 2 0, disintegrate paper with glass rod, and add ca 3 g 
Na 2 2 , keeping beaker well covered with watch glass. Warm 
on steam bath with frequent shaking until all S is oxidized to 
sulfate, adding more Na 2 2 if necessary. Acidify slightly with 
HC1 (1+4), filter to remove shreds of paper, wash thoroly with 
hot H 2 0, and det. S in filtrate as in 920.31B. 

Ref.: JAOAC 3, 353(1920). 



920.34 



Lime Sulfur Solutions 

and Dry Lime Sulfur 

Final Action 



A. Sulfate Sulfur 

Slightly acidify soln from 920.32B with HC1 (1+4) and heat 
to bp. Add slowly, with const stirring, slight excess 10% BaCl 2 
soln, boil 30 min, let stand overnight, and filter. Calc. S from 
wt BaS0 4 , and report as % sulfate S. 

B. Total Calcium 

To 25 mL prepd soln, 920.31A(a) or (b), add 10 mL HC1, 

evap. to dryness on steam bath, add H 2 and few mL HC1 
(1+4), warm until all CaCl 2 dissolves, and filter to remove S 
and any Si0 2 present. Dil. filtrate to 200-250 mL, heat to bp, 
add few mL NH 4 OH in excess, and then add excess satd 
{NH 4 ) 2 C 2 4 soln. Continue boiling until pptd CaC 2 4 assumes 
well defined granular form, let stand 1 hr, filter, and wash few 
times with hot H 2 0. Ignite at 950° in Pt crucible to const wt 
(CaO) and calc. % Ca. CaO x 0.7147 - Ca. 

CAS-7440-70-2 (calcium) 



935.07 Sodium Hypochlorite Solution 

Pesticide Formulations 

Final Action 

Sodium Hypochlorite 

Arsenious Oxide Titration Method 



QAN. Prep, as in 939.12. 
as in 939. 13 A. Stdze against 



A. Reagents 

(a) Arsenious oxide std soln. 

(b) Iodine std soln. — Prep 
(a). 

B. Determination 

Transfer 20 mL sample to 1 L vol. flask and dil. to vol. 
Pipet 50 mL aliquot of mixt. into 200 mL erlenmeyer. Add 
excess As 2 3 soln and then decided excess NaHC0 3 . Titr. ex- 
cess As 2 3 with std I soln, using starch soln, 922.03A(f), or 
the I as its own indicator. Subtract vol. I soln, corrected to 
O.liV, from vol. As 2 3 soln used, and from this value and sp 
gr of soln, calc. % NaOCl. 

1 mL 0. \N As 2 3 = 0.003722 g NaOCl 

Refs.: JAOAC 18, 63(1935). 

CAS-768 1-52-9 (sodium hypochlorite) 



935.08 Sodium Hypochlorite Solution 

Pesticide Formulations 
Final Action 

A. Chlorine (Available) 

Calc. % available CI from titrn, 935.07B. 1 mL 0. \N As 2 3 

- 0.003545 g available CI. 

B. Chlorine (Chloride) 

Pipet 50 mL aliquot prepd soln, 935. 07B, into 200 mL er- 
lenmeyer and add slight excess As 2 3 soln, 935.07A(a), calcd 
from NaOCl titrn; add slight excess HN0 3 , neutze with CaC0 3 , 
and titr. with 0.1/V AgN0 3 , 941.18, using K 2 Cr0 4 soln, 



AOAC Official Methods of Analysis (1990) 



Inorganics and Organometallics 



161 



941.18B(b), or the Ag 3 As0 4 formed in soln, as indicator. Det. 
blank on reagents and correct for any CI found. From this cor- 
rected titrn and sp gr of sample, calc. % CI. From this value 
subtract V2 the % available CI. Difference - % chloride CI. 

CAS-7782-50-5 (chlorine) 

C. Sodium Hydroxide 

Stdze pH meter equipped with calomel and glass electrodes, 
using std pH 6.9 buffer soln, 964.24(d). 

Place 50 mL 10% BaCl 2 .2H 2 soln and 30 mL 3% H 2 2 
soln in 250 mL beaker. Neutze to pH 7.5 with ca 0.1/V NaOH, 
using pH meter. Add 10 mL sample from pipet, stir vigorously 
1 min, and titr. to pH 7.5 with stdzd 0.1/V HC1, using pH 
meter. 

% NaOH - (mL HC1 x normality x 4.0)/ 

(mL sample x sp gr) 

Ref.: JAOAC 18, 63(1935); 43, 346(1960). 

CAS-1310-73-2 (sodium hydroxide) 

D. Carbon Dioxide* 
—Surplus 1965 

See 4.158-4.159, 10th ed. 



B. Total Chlorine 

Dissolve 0.5 g sample in 50 mL H 2 in erlenmeyer and add 
slight excess std As 2 3 soln as calcd from active CI titrn, 
935.10A. Add 5 mL HN0 3 (1+4), neutze with CaC0 3f and 
titr. with std AgN0 3 , 941.18, using K 2 Cr0 4 , 941.186(b), as 
indicator. Det. blank on reagents and correct for any CI found. 
From corrected titrn, calc. % total CI in sample. 1 mL 0.1 AT 
AgN0 3 = 0.003545 g CI. If total C\ exceeds active CI, NaCl 
is indicated. 

C. Sodium* 
—Surplus 1974 

See 6.183, 11th ed. 
Refs.: JAOAC 18, 63(1935). 



927.01 



Unsulfonated Residue 

of Mineral Oats (1) 

Final Action 



A. Reagent 

(Caution: See safety notes on sulfuric acid and fuming acids.) 



935.09 Chlorine (Available) 

in Calcium Hypochlorite 
and Bleaching Powder 

Arsenious Oxide Titration Method 
Final Action 

Weigh 5-10 g thoroly mixed sample into porcelain mortar, 
add 30-40 mL H 2 0, and triturate to smooth cream (high-test 
Ca(OCl) 2 will dissolve readily and not form a cream). Add 
more H 2 0, stir well with pestle, and let insol. residue settle 
few moments. Pour mixt. off into 1 L vol. flask, add more 
H 2 0, and thoroly triturate sample and pour off as before. Re- 
peat operation until all material is transferred to flask. Rinse 
mortar and pestle, catch wash H 2 in flask, dil. to vol., and 
mix. Without letting material settle, pipet 25-50 mL aliquot 
into 200 mL erlenmeyer. Add excess std As 2 3 soln, 
935.07A(a), and then decided excess of NaHC0 3 . Titr. excess 
As 2 3 with std 1 soln, 935.07A(b), using starch soln, 
922.03A(f), or 1 as its own indicator. Subtract vol. I soln, 
corrected to 0. IN, from vol. As 2 3 soln used, and calc. % 
available CI. 1 mL QAN As 2 3 - 0.003545 g available CI. 

Refs.: JAOAC 18, 65(1935). 

CAS-7778-54-3 (calcium hypochlorite) 



935.10 



ChSoramsne T 

Pesticide Formulations 

Final Action 



A. Active Chlorine 

Transfer 0.5 g sample to 300—500 mL erlenmeyer, dissolve 
in 50 mL H 2 0, and add excess std As 2 3 soln, 935.07A(a), 
and 5 mL H 2 S0 4 (1 + 4). Add decided excess NaHC0 3 and titr. 
excess As 2 3 with std I soln, 935.07A(b), using starch soln, 
922.03A(f), or 1 as its own indicator. From this titrn, calc. 
active CI in sample. 1 mL QAN As 2 3 = 0.001773 g active 
CI. (To convert active CI to available CI, multiply active CI 
by 2.) 



Fuming 38N sulfuric acid. — In tared g-s bottle (2.5 L acid 
bottle is convenient) mix fuming H 2 S0 4 (free from N oxides) 
(jc) with H 2 S0 4 (y) to obtain mixed acid (z), contg slightly 
>82.38% total S0 3 . Depending on strength of fuming acid 
available, use following proportions of 2 acids: 100 parts jc 
(15-20% free S0 3 ) to 50 parts y; 100 parts jc (20-30% free 
S0 3 ) to 75 parts y; or 100 parts x (50% free S0 3 ) to 140 parts 
y. Mix thoroly (considerable heat is generated), let cool, and 
again weigh to det. amt mixed acid obtained. Det. exact strength 
of mixed acid (2) and also of reserve supply of acid (y) as 
follows: 

Pour ca 50 mL into small beaker and fill ca 10 mL weighing 
bulb or pipet by slight suction, wiping off outside of bulb with 
moist, then with dry, cloth. Weigh on analytical balance and 
let acid flow slowly down sides of neck of 1 L vol. flask into 
ca 200 mL cold H 2 0. (These sizes of bulb and flask give final 
soln ca 0.57V.) When bulb has drained, wash all traces of acid 
into flask, taking precautions against loss of S0 3 fumes. Dil. 
to vol. and titr. from buret with std alkali, using same indicator 
as used in stdzg. Calc. S0 3 content of both acids, and add 
calcd amt of reserve acid (y) to amt of mixed acid (2) on hand 
to bring z to 82.38% total S0 3 (equiv. to 100.92% H 2 S0 4 ). 
After adding required amt of y, again analyze mixed acid to 
make certain it is of proper concn (±0.15% H 2 S0 4 ). Keep acid 
in small bottles or in special dispenser bottle (2) to prevent 
absorption of H 2 from air. 

B. Determination 

Pipet 5 mL sample into 6" Babcock cream bottle, 938.01 B(b), 
either 9 g 50% or 18 g 30% type. To reduce viscosity of heavy 
oils, warm pipet after initial drainage by passing it several times 
thru flame; then drain thoroly. If greater accuracy is desired, 
weigh measured sample and calc, exact vol. from wt and sp 
gr. Slowly add 20 mL 3SN H 2 S0 4 , gently shaking or rotating 
bottle and taking care that temp, does not rise above 60°. Cool 
in ice-H 2 if necessary. When mixt. no longer develops heat 
on shaking, agitate thoroly, place bottle in H 2 bath, and heat 
10 min at 60-65°, keeping contents of bottle thoroly mixed 
by shaking vigorously 20 sec at 2 min intervals. Remove bottle 
from bath and add H 2 S0 4 until oil is in graduated neck. Centrf . 
5 min (or longer if necessary to obtain const vol. of oil) at 
1200-1500 rpm. Read vol. of unsulfonated residue from grad- 



162 



Pesticide Formulations 



AOAC Official Methods of Analysis (1990) 



uations on neck of bottle and, to convert to mL, multiply read- 
ing from 9 g 50% bottle by 0.1 and reading from 18 g 30% 
bottle by 0.2. From result obtained, calc. % by vol. of un- 
sulfonated residue. 

Refs.: (1) JAOAC 10, 30, 124(1927); 11, 35(1928). 
(2) USDA Bull. 898, p. 48. 



D, Unsulfonated Residues 

Using 5 mL of the recovered oil, 
927.01B. 



926.02B, proceed as in 



926.01 



Soaps 

Final Action 



A. Moisture 

Weigh ca 20 g sample into 300-500 mL flask; add 50 mL 
toluene (tech. grade is satisfactory); and, to prevent foaming, 
add ca 10 g lump rosin (do not use powd). Distil into Dean 
and Stark type distg tube receiver and continue distn until no 
more H 2 collects in receiver. Cool contents of tube to room 
temp., read vol. H 2 under toluene in tube, and calc. % H 2 0. 

Ref.: JAOAC 9, 27(1926). 

B. Sodium and Potassium* 
—Surplus 1974 

Se<? 6.094, 11th ed. 



926.02 Mineral Oil-Soap Emulsions 

Final Action 

A. Water 

Weigh ca 25 g sample and proceed as in 926. 01 A, except 

use less rosin. 

Ref.: JAOAC 9, 28(1926). 

a Total Oil 

Weigh ca 10 g sample into Babcock cream bottle, 
920.111B(a). Dil. with ca 10 mL hot H 2 and add 5-10 mL 
H 2 S0 4 (1 + 1). Heat in hot H 2 bath ca 5 min to hasten sepn 
of oil, add enough satd NaCl soln to bring oil layer within 
graduated neck of bottle, centrf. 5 min at 1200 rpm, and let 
cool. Read vol. of oil layer, det. density, and from these val- 
ues calc. wt and %. From this % value deduct % fatty acids 
(and phenols if present), detd sep., to obtain % oil. 

Ref.: USDA Bur. Chem. Bull. 105, p. 165. 
C. Soap 

(Error will result if apparent mol. wt of fatty acids varies ap- 
preciably from that of oleic acid.) 



Weigh 20 g sample into separator, add 60 mL pet ether, and 
ext mixt. once with 20 mL and 4 times with 10 mL 50% al- 
cohol. Break emulsion if necessary by letting 1 or 2 mL 20% 
NaOH soln run down wall of separator. Then gently swirl sep- 
arator and let stand few min. Drain ale. layers and wash suc- 
cessively thru pet ether contained in 2 other separators. Com- 
bine ale. exts in beaker and evap. on steam bath to remove 
alcohol. Dissolve residue in ca 100 mL H 2 made alk. with 
NaOH. Transfer to separator, acidify with HO or H 2 S0 4 , ext 
3 times with Et ether, and wash ether exts twice with H 2 0. 
Combine ether exts, evap. in weighed beaker on steam bath, 
and weigh as fatty acids. From wt fatty acids, calc. % soap 
in sample as Na or K oleate. 

Ref.: JAOAC 9, 28(1926). 



E. Ash 

Evap. 10 g sample, or more if necessary, in Pt dish. Ignite, 
and leach charred mass with H 2 0. Ignite residue, add teach- 
ings, evap. to dryness, ignite, and weigh. From this wt, calc. 
% ash. Test ash for Cu, Ca, CaF 2 , etc. 

Ref.: JAOAC 9, 29(1926). 



930.13* Mercury in Organic Mercurial 

Seed Disinfectants 
Volatilization Method 

Final Action 
Surplus 1965 



See 4.150-4.151, 10th ed. 



930.14* Mercury in Organic Mercurial 

Seed Disinfectants 
Precipitation Method 

Final Action 
Surplus 1974 

See 6.173, 11th ed. 



971.04 Mercury in Organic Mercurial 

Seed Disinfectants 
Titrimetric Method 

First Action 1971 
Final Action 1974 

A. Principle 

Sample is digested under H 2 0-cooled condenser with fum- 
ing H 2 S0 4 -fuming HN0 3 . Hg is detd by titrn with std SCN 
soln with ferric alum as indicator. Small amts of chloride are 
oxidized to CI and expelled thru condenser. Not applicable in 
presence of large amts of Cl-contg materials. 

3. Reagents 

(a) Ferric indicator. — Dissolve 8 g FeNH 4 (S0 4 ) 2 .12H 2 in 
80 mL H 2 0. Add enough HN0 3 to destroy brown Fe color and 
dil. to 100 mL with H 2 0. 

(b) Ferrous sulfate soln. — Dissolve 1 g FeS0 4 .7H 2 in H 2 0, 
add 1 mL H 2 S0 4 , and dil. to 100 mL with H 2 0. Prep, fresh 
for each detn. 

C. Preparation of Sample 

(a) Solns. — Mix thoroly and weigh, by difference, amt 
sample (max. 10 g) contg preferably 0.07 g Hg into 500 mL 
¥ erlenmeyer. 

(b) Dusts. — Mix thoroly and, using glass weighing dish, 
weigh amt sample as in (a). Transfer thru powder funnel into 
500 mL ¥ erlenmeyer. 

D. Determination 

{Caution: Conduct detn in well ventilated hood. Method is 

dangerous in presence of material which reacts violently with 

H 2 S0 4 and/or HN0 3 , See safety notes on wet oxidation, nitric 

acid, sulfuric acid, fuming acids, and mercury salts.) 



AOAC Official Methods of Analysis (1990) 



Inorganics and Organometalucs 



163 



Connect straight-tube, H 2 0-cooled condenser to erlenmeyer 
contg sample. Place flask in cold H 2 bath. Carefully add 10 
mL H 2 S0 4 thru top of condenser and mix by swirling. Add in 
small portions, swirling after each addn, 30—40 mL fuming 
H 2 S0 4 (20% free S0 3 ) thru top of condenser, followed by 10 
mL red fuming HN0 3 (98% HN0 3 ). Remove from bath and 
dry outside of flask. Heat with small flame to reflux at ca 30 
drops /min with red fumes persisting in flask and condenser. 
Heat 30 min; if small amt chloride is present, heat 2 hr with 
occasional addn of fuming HN0 3 as required. Cool, and add 
100 mL cold H 2 slowly thru top of condenser while cooling 
flask in cold H 2 bath. Add 2 or 3 glass beads or boiling chips 
and boil until N oxides have been expelled to top of condenser 
(ca 2 min). Wash condenser with 50 mL cold H 2 0, disconnect 
flask, and add satd KMn0 4 soln until color remains purple. (If 
large amts insol. material are present, filter hot soln thru me- 
dium tight asbestos mat in gooch before addn of KMn0 4 . Wash 
flask and filter 5 times with hot H 2 0, and then add KMn0 4 .) 
Cool flask, and destroy KMn0 4 with fresh 1% FeS0 4 soln. 
Add 10 mL ferric indicator and titr. with 0.17V NH 4 SCN or 
KSCN, 942.26, to appearance of first permanent faint orange. 
1 mL 0AN NH 4 SCN or KSCN = 0.01003 g Hg. 

Ref.: JAOAC 54, 685(1971). 

CAS-7439-97-6 (mercury) 



973.11 Mercury in Organic Mercurial 

Seed Disinfectants 
Gravimetric Method 

First Action 1973 
Final Action 1975 

AOAC-CIPAC Method 

(Applicable in presence of large amts CLcontg materials; not 
applicable to chioro- or nitrophenols nor to materials not de- 
composed by digestion mixt.) 

A. Reagents 

(a) Dilute sulfuric acid.— Add 30 mL H 2 S0 4 to H 2 in 100 
mL vol. flask, cool, and dil. to vol. with H 2 0. 

(b) Sodium sulfite soln. — 10%. Dissolve 10 g Na 2 S0 3 in 
H 2 in 100 mL vol. flask and dil. to vol. with H 2 0. 

(c) Ammonium citrate soln. — Should have sp gr of 1.09 at 
20° and pH of 7.0 as detd potentiometrically. 

Dissolve 370 g cryst. citric acid in 1.5 L H 2 and nearly 
neutze by adding 345 mL NH 4 OH (28-29% NH 3 ). If concn 
of NH 3 is 28%, add correspondingly larger vol. and dissolve 
citric acid in correspondingly smaller vol. H 2 0. Cool, and check 
pH. Adjust with NH 4 OH (1 + 7) or citric acid soln to pH 7. 
Dil. soln, if necessary, to sp gr of 1.09 at 20°. (Vol. will be 
ca 2 L.) Keep in tightly stoppered bottles and check pH from 
time to time. If pH has changed from 7.0, readjust. 

(d) Precipitating reagent. — Add 20 mL 1,2-propanedi- 
amine (Eastman Kodak Co., P3170) to 100 mL \M CuS0 4 
soln. Store in g-s container. 

(e) Wash soln. — Add 1 g KI and 2 mL pptg reagent to 1 
L H 2 0. 

B. Preparation of Sample 

(a) Solns. — Mix thoroly and weigh, by difference, sample 
(max. 5 g) contg 0.02-0.08 g Hg into 125 mL ¥ erlenmeyer. 

(b) Dusts. — Mix thoroly and, using glass weighing dish, 
weigh sample as in (a). Transfer thru powder funnel into 125 
mL ¥ erlenmeyer. 



C. Determination 

(Caution: Conduct detn in well ventilated hood.) 

Add to sample in following order: 5 mL ethylene glycol, 
swirling to thoroly suspend solids, 4 g KI, 10 mL dil. H 2 S0 4 , 
0.4 g I, and 2 glass beads. After thoro mixing, connect straight- 
tube, H 2 0-cooled condenser and, with low flame, heat to slight 
boil so that liq. condenses in lower portion of condenser. Swirl 
occasionally, avoiding excessive heat and crystn of large amt 
I in condenser. Reflux 1 hr and, while cooling flask in H 2 
bath, immediately wash warm condenser with heavy stream of 
ca 25 mL H 2 0. (If dye or I persists in condenser, loosen by 
reheating flask contents, without H 2 in condenser, until liq. 
refluxes slightly beyond adhering material. Wash condenser 
again with ca 25 mL H 2 0, and cool flask.) Disconnect con- 
denser and wash connections directly into flask. Add ca 2 mL 
10% Na 2 S0 3 dropwise, with swirling, until I color slightly 
lightens. (Excess I must be present.) Neutze soln with NH 4 OH, 
using pH test paper, until very slightly alk. (pH 7.0-7.3). Cool, 
and filter with vac. thru retentive paper (S&S Blue Ribbon, 
or equiv.) in buchner into 400 mil beaker. Wash flask and 
paper thoroly, keeping total filtrate <150 mL. Add 50 mL 
NH 4 citrate soln, bring mixt. just to bp, and stir in 5 mL pptg 
reagent. Cool and let stand >2 hr (preferably overnight); filter 
thru medium porosity glass crucible, previously dried at 105° 
and weighed. Transfer ppt with wash soln, and wash with same 
soln several times. Rinse I from ppt with ca 25 mL alcohol in 
5 mL portions (some samples may require up to 50 mL) until 
filtrate is colorless. (Let alcohol stand few min with occasional 
swirling after each addn before applying suction. Ppt should 
be suspended in liq. each time.) Wash ppt with three 5 mL 
portions CHC1 3 , suspending ppt each time as above until dye 
and pesticides are completely removed. Finally wash with 5 
mL alcohol, dry 30 min at 105°, cool, and weigh. 



Ref. 



Wt Hg = wt ppt X 0.218 
JAOAC 56, 572(1973); 58, 309(1975). 



CAS-7439-97-6 (mercury) 



948.03* Alpha-Naphthylthiourea 

in Rodenticide Formulations 

First Action 
Surplus 1965 

See 4.132, 10th ed. 



939.01 Thalious Sulfate 

in Rodenticide Formulations 

Final Action 

(Caution: See safety notes on asbestos, wet oxidation, nitric 
acid, fuming acids, and pesticides.) 

Weigh sample contg 0.1-0.15 g T1 2 S0 4 (usually 10 g), 
transfer to 800 mL Kjeldahl flask, and add 25 mL H 2 S0 4 fol- 
lowed by 5-10 mL HN0 3 . After first violent reaction ceases, 
heat until white fumes of H 2 S0 4 appear. Add few drops fuming 
HN0 3 and continue heating and adding HNO3 until org. matter 
is destroyed, as shown by colorless or light yellow soln. Cool, 
add 10-15 mL H 2 0, again cool, and wash contents of flask 
into 400 mL beaker, continuing washing until vol. is 60-70 
mL. Boil several min to remove all HN0 3 , cool, and filter into 
400 mL beaker. Wash with hot H 2 until vol. in beaker is 175 



164 



Pesticide Formulations 



AOAC Official Methods of Analysis (1990) 



mL, neutze with NH 4 OH, and then slightly acidify with H 2 S0 4 
(1+4). Add 1 gNaHS0 3 to ensure reduction of thallic to thal- 
lous state. Heat to bp, add 50 mL 10% KI soln, stir, and let 
stand overnight. Filter thru tight gooch contg 2 disks S&S 589 
white ribbon paper covered by medium pad of asbestos. Wash 
4 or 5 times with 10 mL portions 1% KI soln, and finally with 
absolute alcohol. Dry to const wt at 105° (1-1.5 hr), and weigh 
as TIL 

% T1 2 S0 4 = (g Til x 0.7619 x 100)/g sample 

Refs.: JAOAC 22, 411(1939); 25, 79(1942); 28, 72(1945). 

CAS-7446-18-6 (thallous sulfate) 



966.05 Fumigant Mixtures 

Gas Chromatographic Method 

First Action 1966 
Final Action 1985 

(Applicable to org. components of CS 2 , CC1 4 , (CH 2 )2C1 2 , and 
(CH 2 )2Br 2 mixts. Precautions: Handle with care in hood or 
well-ventilated area. Mixts are volatile, poisonous, and some- 
times flammable and may be fatal if inhaled or swallowed. 
They cause skin and eye irritation. In case of contact, im- 
mediately remove contaminated clothing and flush affected area 
with copious amts of H 2 0. Do not reuse clothing until free of 
contamination. Do not use containers or equipment of Al, Mg, 
or their alloys.) 

A. Principle 

Components are detd by GC. Peak area of each component 
is measured and compared to stds of same fumigant mixt. Pre- 
cision of method is ±0.6% for each component. 

B. Sampling 

Obtain representative 1 L sample from container. Sample 
bulk containers by means of weighted bottle, lowered toward 
bottom and raised at such rate that it is 3 / 4 full when with- 
drawn. Sample drums or small containers with thief or thru 
tap or valve located so that sample comes from well below 
surface. Prevent contamination of product or sample. 

Place sample in clean, dry, and sol v. vapor-tight glass bottle 
of such size that it is nearly filled (not above shoulder) by 
sample. Vapor-tight g-s bottles or screw-cap bottles with Sn 
foil lined caps are satisfactory. Store samples at low temp.; 
cool to <18° before opening for analysis. 

C. Apparatus 

(a) Gas chromato graph. — With flame ionization or thermal 
conductivity detector. Typical operating conditions: Column 
temp., 110°; injection port temp., 200°; flow rate, 80 mL He/ 
min. 

(b) Recorder. — 0.05-1.05 mv, full scale response. Inte- 
grator may be used. 

(c) Syringe. — Hamilton Co. 10 jjlL No. 701N, or equiv. 

(d) Column. — 1.2 m (4') stainless steel, 7 4 " od, 0.194" id, 
packed with reagent 966.05D(a). Max. temp, is 160°. Other 
columns can be used but chromatgc conditions and sample size 
must be adjusted in accordance with column requirements. One 
such column is: 3 m (10') stainless, 3 / J6 " od, 0.12" id, packed 
with 20% by wt A^,A^~bis-(2-cyanoethyl) formamide on 80-100 
mesh Chromosorb W, acid-washed. Columns are available from 
com. suppliers. Criterion for use is emergence of each com- 
ponent of mixt. of CS 2 , CC1 4 , (CH 2 ) 2 C1 2 , and (CH 2 ) 2 Br 2 as 
sep. peak. 



D. Reagents 

(a) Column packing. — 30% by wt tricresyl phosphate on 
Chromosorb P, 30-60 mesh. 

(b) Carbon disulfide std. — ACS. 

(c) Carbon tetrachloride std. — ACS. 

(d) Ethylene dichloride std. — Purified 1,2-dichloroethane, 
available from laboratory supply houses, or use center cut of 
fractionation of com. product. 

(e) Ethylene dibromide (1 ,2-dibromoethane) std. — Puri- 
fied or distd as in (d). 

E. Preparation of Standards 

Prep, fresh stds just before analysis which approximate ex- 
pected composition, by wt, of fumigant mixt. Place proper wt 
of each component in 25 mL g-s vol . flask and mix well . Do 
not prep, by vol. Cool CS 2 to prevent loss. Adjust wt stds to 
detector response. 

Carefully fill weighed 10 mL vol. flask to mark with prepd 
std and weigh. Use this wt to det. g/5 |ulL values for each 
component of std. 

F. Determination 

Purge column thoroly at 110° before use. Establish base- 
line at full sensitivity. Inject 5 \xL std fumigant mixt. into 
chromatograph. Attenuate successively so that each peak is at 
max. % of chart scale, adjusting sample size and attenuation, 
if necessary. Repeat injection. Detd area for each component, 
corrected for any baseline drift, should differ by <1%. Order 
of elution from column is: CS 2 , CC1 4 , (CH 2 ) 2 C] 2 , and (CH 2 ) 2 Br 2 . 
Total analysis time is ca 21 min. 

Inject 5 |iL sample into chromatograph. Det. corrected area 
of each component from chromatogram, or note integrator 
reading. 

g Components X C/B, where S — wt component in std, 
B — area for component in std, and C = area for component 
in sample. Perform calcn for each component in sample. 

% Component = g component in sample x 100/ sum of g 
components in sample 

Last equation is not applicable in presence of unmeasured 
contaminants. 

Refs.: JAOAC 48, 576(1965); 49, 207(1966). 

CAS-75-15-0 (carbon disulfide) 
CAS-56-23-5 (carbon tetrachloride) 
CAS- 106-93-4 (ethylene dibromide) 
CAS- 107-06-2 (ethylene dichloride) 



PESTICIDES RELATED TO NATURAL 
PRODUCTS AND THEIR SYNERGISTS 



953.05 Allethrin (Technical) 

and Pesticide Formulations 

Titrimetric Method 
First Action 

{Caution: See safety notes on on pesticides.) 

A. Principle 

Allethrin reacts quant, with ethylenediamine to form chry- 
santhemum monocarboxylic acid which is detd by titrn with 
std NaOMe in pyridine. Chrysanthemum monocarboxylic acid, 
anhydride, and acid chloride interfere quant, and are detd in- 
dependently . 



AOAC Official Methods of Analysis (1990) 



Synthetics Related to Natural Products 



165 



B. Reagents 

(a) Absolute alcohol. — SDF No. 2-B is satisfactory. 

(b) Methanolic hydrochloric acid std soln. — 0. 17V. Dil. 17 
ml HC1 (1 + 1) to 1 L with anhyd. MeOH. Stdze against std 
OAN NaOH, using phthln. If used at temp,, T, different from 
that at which stdzd, T (h calc. corrected normality = N[\~0.00\(T 

- r )]. 

(c) Sodium methylate std soln. — 0.17V in pyridine. Transfer 
50 mL 2N NaOMe (Caution: See safety notes on sodium bi- 
phenyl, methylate, and ethyl ate.) to 1 L bottle contg 75 mL 
anhyd. MeOH and dil. to 1 L with redistd pyridine. Stdze against 
NBS benzoic acid, using pyridine as solv. and thymolphthal- 
ein, (i), as indicator. Dispense from 50 mL automatic buret 
with vents connected to Ascarite tubes. Stdze daily against std 
methanolic HCl, (b). 

(d) Methanolic potassium hydroxide std soln. — 0.027V. 
Dissolve 112 g KOH in 1 L MeOH. Stdze as in 926.16D. 

(e) Morpholine soln. — Transfer 8.7 mL redistd morpholine 
to 1 L bottle and dil. to 1 L with anhyd. MeOH. Fit bottle 
with 2-hole rubber stopper; thru 1 hole insert 20 mL pipet so 
that tip extends below surface of liq. , and thru other hole insert 
short piece of glass tubing to which is attached aspirator bulb. 

(f) Ethylenediamine. — Redistd com. grade contg <3% H 2 0. 
Dispense from automatic buret with vents connected to As- 
carite tubes. 

(g) Dimethyl yellow -methylene blue mixed indicator, — Dis- 
solve 1 g dimethyl yellow (p-dimethylaminoazobenzene; cau- 
tion', see safety notes on carcinogens) and 0.1 g methylene blue 
in 125 mL anhyd. MeOH. 

(h) a-Naphtholbenzein indicator. — 1% ale. soln. 
(i) Thymolphthalein indicator. — 1% pyridine soln. 

C. Determination of Chrysanthemum Monocarboxylic Acid 
Chloride 

Add 8-10 drops mixed indicator, (g), to ca 150 mL anhyd. 
MeOH and add 0.17V HCl, (b), dropwise until soln appears 
reddish brown by transmitted light. Add 0.027V KOH, (d), 
dropwise until appearance of first green. Transfer 25 mL to 
each of three 125 mL g-s erlenmeyers, reserving 1 flask as 
ref. color for end point. Into each of other flasks add 1.5-2.5 
g sample from weighing pipet, swirling flask while adding 
sample. Within 5 min, titr. with 0.027V KOH, (d), to first green 
end point, using blank as ref. color. Calc. milliequiv. chry- 
santhemum monocarboxylic acid chloride/g sample, C — V 
X 7V/g sample, where V= mL TV normal KOH required; 

% Chrysanthemum monocarboxylic acid chloride — C x 18.67 

D. Determination of Chrysanthemum Monocarboxylic Acid 

Transfer 25 mL anhyd. alcohol to each of two 125 mL g-s 
erlenmeyers, add 8-9 drops a-naphtholbenzein indicator, and 
cool to 0° in ice bath. Neutze by adding 0.02/V NaOH dropwise 
to bright green end point. To each flask add 1.5-2.5 g sample 
from weighing pipet. Immediately titr. with 0.027V NaOH, 
936. 16C, to first bright green end point. Calc. milliequiv. 
chrysanthemum monocarboxylic acid and acid chloride/g 
sample: D = X x 7V/g sample, where X = mL TV normal NaOH 
required; (D - C) x 16.82 = % chrysanthemum monocar- 
boxylic acid. 

E. Determination of Chrysanthemum Monocarboxylic 
Anhydride 

Pipet 20 mL morpholine soln, (e), into each of four 250 mL 
erlenmeyers, using same pipet. Fill pipet by exerting pressure 
in bottle with aspirator bulb. Reserve 2 flasks for blanks; into 
each of other flasks add 1 .5-2.5 g sample from weighing pi- 
pet. Swirl flasks and let samples and blanks stand 5 min at 
room temp. Add 4-5 drops mixed indicator, (g), to each flask 



and titr. with OAN HCl, (b), until color changes from green 
to faint red when viewed by transmitted light. Calc. millie- 
quiv. chrysanthemum monocarboxylic anhydride /g sample: E 
= (B - Y) x TV/g sample, where Y = mL TV normal HCl 
required for sample, and B — mL TV normal HCl required for 
blank; (E — 2C) x 31.84 = % chrysanthemum monocarbox- 
ylic anhydride. 

F. Determination of Allethrin 

Add sample contg 0.8—1.1 g allethrin to each of two 250 
mL erlenmeyers from weighing pipet. To each of 2 flasks as 
blanks and to samples add 25 mL ethylenediamine, (f), with 
swirling. Let samples and blanks stand 2 hr at 25±2°. Wash 
down sides of flasks with 50 mL redistd pyridine. To each 
flask add 6-10 drops thymolphthalein indicator, (i), and titr. 
with OAN NaOMe, (c), to first permanent blue-green end point. 
(With colorless samples, first blue end point may be used.) 
Calc. milliequiv. allethrin /g sample: F — (Z - B) xTV/g sam- 
ple, where Z = mL TV normal NaOMe required for sample, 
and B = av. mL TV normal NaOMe required for blank; (F + 
C - D - E) x 30.24 = % allethrin. 

Refs.: Anal. Chem. 25, 1207(1953). JAOAC 40, 732(1957). 

CAS-37-98-4 (allethrin) 



973.1 2 d-frans-Allethrin 

in Pesticide Formulations 
Gas Chromatographic Method 

First Action 1973 
Final Action 1978 

(Caution: See safety notes on pesticides.) 

A. Principle 

d-trans- Allethrin is dild in acetone contg dibutyl phthalate 
as internal std. Ratios of GC peak hts of d-trans-allethrin and 
dibutyl phthalate in sample and std are compared for quant, 
detn. Method is applicable to both tech. d-trans-eulcthrin and 
various formulations of it. Not applicable to formulations contg 
large amt MGK Repellent 874 (2-hydroxyethyl-n-octyl sul- 
fide). 

B. Apparatus and Reagents 

(a) Gas chromato graph. — Equipped with flame ionization 
detector and 1.2 m (4') x 4 mm id glass column packed with 
5% OV-1 (Analabs, Inc.) on 80-100 mesh Chromosorb W 
(HP). Operating conditions: temps (°) — column 165, injection 
port 230, detector 230; gas flows (mL/min) — N carrier gas 
125, air 350-400, H 40-50; sensitivity— 10" 9 amp full scale, 
attenuation 4x for tech. material, 10~ 9 amp full scale, atten- 
uation 1 for formulations. Before use, condition column 2-3 
hr at 275° with N flow 50 mL/min. If necessary, vary column 
temp, or gas flow to attain retention times of ca 4 and 7 min 
for internal std and d-trans- allethrin, resp. Also vary detector 
sensitivity or injection vol. to attain >100 mm peak ht for each 
compd (ca 16 |xg d-trans -allethrin). Theoretical plates /ft must 
be >200. 

Calc. theoretical plates/ft (N) as follows: N = 16L 2 /(M 2 x 
F), where L = retention of GC peak in mm; M = peak baseline 
produced by drawing tangents to points of inflection of peak; 
and F = length of column (ft). 

(b) Internal std soln. — 4.0 mg dibutyl phthalate /mL ace- 
tone. 

(c) d-trans-Allethrin std solns. — (J) Soln 1 . — Approx. 4 mg/ 
mL. Accurately weigh ca 1.0 g d-trans-a\lethrin (available from 
McLaughlin Gormley King Co., 8810 Tenth Ave N, Minne- 



166 



Pesticide Formulations 



AOAC Official Methods of Analysis (1990) 



apolis, MN 55427) into 50 mL vol. flask and dil. to vol. with 
acetone. Pipet 20 mL this soln into 100 mL vol. flask, add 50 
mL internal std soln by pipet, and dil. to vol. with acetone. 
Use this soln for detn of tech. material. (2) Soln 2. — Approx. 
1 mg/mL. Pipet 25 mL Soln J into 100 mL vol. flask and dil. 
to vol. with acetone. Use this soln for detn of d-trans-ailethrin 
in formulations. 

C. Preparation of Sample 

(a) Technical material. — Accurately weigh sample contg 
ca 1.0 g d-trans-a\\ethrin into 50 mL vol. flask and dil. to vol. 
with acetone. Pipet 20 mL aliquot into 100 mL vol. flask, add 
50 mL internal std soln by pipet, and dil. to vol. with acetone. 

(b) Formulations. — Accurately weigh sample contg ca 200 
mgd-rratt.y-allethrin into 50 mL vol. flask, add 25 mL internal 
std soln by pipet, and dil. to vol. with acetone. Pipet 25 mL 
aliquot into 100 mL vol. flask and dil. to vol. with acetone. 

D. Gas Chromatography 

(a) Technical material. — Inject aliquots (ca 3 |xL) std Soln 
J until ratio of d- trans -a\\othrin:dibuty\ phthalate peak hts var- 
ies <1% for successive injections. Repeat with sample soln, 
followed by duplicate injections of std soln. If peak ht ratios 
differ >± 1% from previous std injections, repeat series of in- 
jections. 

(b) Formulations. — Proceed as in (a), using std Soln 2. Re- 
peat std injections after each series of 3 sample injections. If 
peak ht ratios differ >±1.5% from previous std injections, 
repeat injections. 

E. Calculations 

(a) Technical material, — Calc. peak ht ratios for duplicate 
std injections before and after sample injections and average 
the 4 values. Calc. and average peak ht ratios for sample in- 
jections. 

% d-trans-Attethrin = (R/R r ) x (W /W) x P 

where W and W = g std and sample, resp.; P = % purity of 
std; and R' and R - peak ht ratios of std and sample, resp. 

(b) Formulations . — Calc. av. for all std peak ht ratios and 
for sample peak ht ratios. 

% rf-fra/w-AUethrin = 2 (R/R r ) x (W /W) x (P/5) 

Ref.: JAOAC 55, 907(1972). 

CAS- 1 37-98-4 (allethrin) 



985.03 Cypermethrin in Pesticide Formulations 

Capillary Gas Chromatographic Method 

First Action 1985 
Final Action 1987 

(Method is suitable for tech. and formulated cypermethrin.) 

A. Principle 

Sample is dissolved in CH 2 Cl 2 contg dicyclohexyl phthalate, 
and 1.0 jjlL is injected into capillary GC in split mode, with 
flame ionization detection. Peak areas are measured for each 
cypermethrin isomer and dicyclohexyl phthalate and compared 
with those from std injection. 

B. Apparatus 

(a) Capillary gas chromato graph. — With heated, glass-lined 
split mode injection port, flame ionization detector, and au- 
tomatic sample injector. Temps (°) — column 240, injection port 
250, detector 250; gas flows (mL/min) — He carrier gas 2.75, 
split vent 200 (split ratio 72.6:1), septum purge 0.5-1.0, He 
auxiliary gas to detector 30, H 60, air 240; column head pres- 



sure 15-20 psig; sample size 1.0 jjlL; retention times (min) — 
cypermethrin isomers: cis A, 11.18, trans C, 11.55, cis B, 
11.85, trans D, 12.02; internal std, 5.58. Adjust parameters 
to assure sepn of 4 peaks and peak hts ca 60-80% full scale 
on chart at quoted retention times. Sepn of isomer peaks is 
critical to avoid inclusion of impurity peaks in active ingre- 
dient calcn. 

(b) Column. — 25 m x 0.32 mm (id) fused silica column 
with thick film OV-1 phase (Hewlett-Packard Co., Avondale 
Division, Cat. No. 19091-62025). Precondition 1 h at 260° 
before use. 

C. Reagents 

(a) Dicyclohexyl phthalate internal std soln. — Weigh 0.9 g 
dicyclohexyl phthalate (Pfaltz and Bauer, 172 E Aurora St, 
Waterbury, CT 06708), dissolve in CH 2 C1 2 , and dil. to 500 
mL. Check internal std soln for interfering components by in- 
jecting 1.0 |xL into chromatography Store in tightly capped 
bottles to avoid evapn. 

(b) Cypermethrin std soln. — Accurately weigh ca 100 mg 
cypermethrin std of known purity (ICI Americas, Inc.) into 
scintillation vial. Pipet 10.0 mL internal std soln into vial, cap, 
and shake to dissolve. Store in tightly capped bottles to avoid 
evapn . 

D. Determination 

(a) Liquid and technical samples. — Accurately weigh amt 
sample contg ca 100 mg cypermethrin into vial. Pipet 10.0 mL 
internal std soln into vial, cap, and shake to dissolve, 

(b) Powder formulations. — Accurately weigh amt sample 
contg ca 100 mg cypermethrin into vial. Pipet 10.0 mL inter- 
nal std soln into vial, cap, and shake on wrist- action shaker 
10 min. Let insoluble inerts settle 10 min before analysis. 

Inject 2 or more 1.0 jxL aliquots of std soln to optimize 
instrument and integration parameters and to stabilize instru- 
ment. Monitor response factor until results agree ±2%. Inject 
4 aliquots of std soln and 2 aliquots of sample in succession. 
Calc. response factor, R, for each injection; and take means 
for std and sample for calcn: 

R = total area of 4 cypermethrin 

isomer peaks/area internal std peak 

Cypermethrin, % = (R/R f ) X (W /W) x P 

where R and R f — response factors for sample and std solns, 
resp.; W and W = mg std and sample, resp.; and P = purity 
(%) of std. 

Ref.: JAOAC 68, 592(1985). 

CAS-523 15-07-8 (cypermethrin) 



986.02 Cypermethrin 

in Pesticide Formulations 

Gas Chromatographic Method 

First Action 1986 

CIPAC-AOAC Method 

A. Principle 

Sample is dissolved in methyl isobutyl ketone contg di- 
(ethylhexyl) phthalate as internal std; 4 cypermethrin isomers, 
isolated as one peak, are detd by gas chromatgy with flame 
ionization detection. 

0. Apparatus and Reagents 

(a) Gas chromatograph with recorder and integrator. — With 
flame ionization detector and 1.0 m X 4 mm (id) glass column 
packed with 3% OV-101 on 100-120 or 80-100 mesh Chro- 



AOAC Official Methods of Analysis (1990) 



Synthetics Related to Natural Products 167 



mosorb WHP, capable of on-column injection. Condition newly 
packed column overnight at 260° with low N flow. Operating 
conditions: temps— inlet 250°, column 230-240°, detector 250°; 
carrier gas flow to elute internal std at ca 5.5 min and cyper- 
methrin at ca 11.5 min with > 30 mm between intercepts of 
tangents on baseline of std and internal std peaks; adjust H and 
air for detector as recommended by manuf . ; adjust sensitivity 
to give peak hts 75% full scale. 

(b) Di-(2-ethythexyl) phthalate (DEHP; dioctyl phthal- 
ate). — Fisher Scientific Reagent, or equiv. 

(c) Methyl isobutyl ketone (MIBK),— GC quality (J.T. Baker 
Inc., no. 9212, or equiv.) 

C. Preparation of Standards 

(a) Internal std soln. — 20 mg DEHP/mL. Weigh ca 10 g 
DEHP into 500 mL vol. flask, dil. to vol. with MIBK, and 
mix (soln I). Concn may be varied to accommodate column 
and instrument differences. If necessary, adjust concn so that 
peak ht or area of DEHP closely matches peak ht or area of 
cypermethrin within 10%. 

(b) Cypermethrin std soln. — 4.0 mg/mL. Warm sealed bot- 
tle of cypermethrin std (ICI- Americas, Inc.) at 40-50° until 
no crystals remain; shake bottle. Accurately weigh, in dupli- 
cate, ca 0.2 g std into 50 mL vol. flask, and dissolve in 3-4 
mL MIBK. Pipet 10.0 mL internal std soln into each flask, 
dil. to vol. with MIBK, and mix (solns CA, CB). Similarly, 
weigh ca 0.1 g cypermethrin std into 25 mL vol. flask, dil. to 
vol. with MIBK, and mix (soln CO). 

D. Preparation of Sample 

(a) Technical formulations . — Proceed as above under cy- 
permethrin std soln, using sample wt contg ca 0.2 g cyper- 
methrin (solns SA, SB, SO). 

(b) Wettable powders. — Accurately weigh, in duplicate, 
sample contg 0.2 g cypermethrin into 50 mL vol. flask, pipet 
in 10.0 mL internal std soln. and add sufficient MIBK to sus- 
pend powder. Thoroly shake flask 10 min, dil. to vol. with 
MIBK, and let powder settle or centrf. until clear. Similarly, 
prep, soln without internal std, using sample contg ca 0.1 g 
cypermethrin /25 mL MIBK. 

(c) Ultra-low volume formulations . — Proceed as above un- 
der technical formulations, beginning "Accurately weigh. . .". 

(d) Emulsifiable concentrates. — Proceed as above under 
wettable powders. 

E. System Performance Check and Determination 

Using instrument conditions listed under Apparatus and Re- 
agents, inject 1.5 |ulL portions of solns I, CO, and SO onto 
column and check for interfering peaks. On-column injection 
is necessary. Inject std soln CA and adjust parameters to give 
peak ht ca 75% full scale with peak quality and elution time 
specified. 

Inject 1.5 jxL portions of std solns CA and CB until response 
ratio (area cypermethrin peak/area internal std peak) varies 
<0.5% of mean. (Area measurements by digital electronic in- 
tegration are preferred over other methods.) Carry out injec- 
tions of std and sample solns in following sequence: CA, , SA 1? 
SA 2 , CBj, CA 2 , SB 1; SB 2 , CB 2 . Average response ratios for 
sample and stds that bracket each sample. Successive response 
ratios should agree ± 5% of their mean. If not, repeat analysis. 

Cypermethrin, % = (R/R') x (W'/W) x P 

where R and R' = av. peak area ratios for sample and std, 
resp.; W = g cypermethrin in std soln; W — g sample extd 
for analysis; and P = % purity of std. 

Ref.: JAOAC 70, 51(1987). 

CAS-523 15-07-8 (cypermethrin) 



986.03 Permethrin in Pesticide Formulations 

Gas Chromatographic Method 

First Action 1986 
Final Action 1988 

CIPAC-AOAC Method 

A. Principle 

Sample is dissolved in methyl isobutyl ketone contg n-oc- 
tacosane as internal std, and permethrin is detd as total area 
of 2 isomer peaks by gas chromatgy with flame ionization de- 
tection. 

B. Apparatus and Reagents 

(a) Gas chromatograph with recorder and integrator. — With 
flame ionization detector and 1.0 m X 4 mm (id) glass column 
packed with 3% OV-210 on 100-120 or 80-100 mesh Chro- 
mosorb WHP, capable of on-column injection. Condition newly 
packed column overnight at 275° with low N flow. Operating 
conditions: temps — inlet 260°, column 190-220°, detector 250°; 
carrier gas flow to elute internal std in ca 4.0 min and trans- 
permethrin at ca 9.5 min with >30 mm between intercepts of 
tangents on baseline of internal std and std cis- and trans-iso- 
mer peaks; adjust H and air for detector as recommended by 
manuf.; adjust sensitivity to give peak hts 75% full scale. 

(b) n-Octacosane std. — With no peaks at retention times of 
permethrin isomers (Kodak Laboratory Chemicals, or equiv.). 

(c) Methyl isobutyl ketone (MIBK). — GC quality (J.T. Baker 
Inc., no. 9212, or equiv.). 

C. Preparation of Standards 

(a) Internal std soln. — 1 mg w-octacosane/mL. Weigh ca 
0.5 g w-octacosane into 500 mL vol. flask, dissolve in 300 mL 
MIBK, dil. to vol. with MIBK, and mix (soln I). Concn may 
be varied to accommodate column and instrument differences. 
If necessary, adjust concn so that peak ht of n-octacosane closely 
matches peak ht of permethrin isomers. 

(b) Permethrin std soln. — 4.0 mg/mL. Warm sealed bottle 
of permethrin std (ICI Americas, Inc.) at 40-50° until no crys- 
tals remain; shake bottle. Accurately weigh, in duplicate, ca 
0.1 g std into 100 mL g-s erlenmeyer. Pipet 25.0 mL internal 
std soln into each flask and shake until permethrin is dissolved 
(solns CA, CB). Similarly, weigh ca 0.1 g permethrin std into 
25 mL vol. flask, dissolve in 15 mL MIBK, dil. to vol. with 
MIBK, and mix (soln CO). 

D. Preparation of Sample 

(a) Technical formulations . — Proceed as above under per- 
methrin std soln, using sample wt contg ca 0.1 g permethrin 
(solns SA, SB, SO). 

(b) Wettable and dustable powders (suspendibility >50%). — 
Accurately weigh, in duplicate, sample contg 0. 1 g permethrin 
into 100 mL g-s erlenmeyer. Pipet 25.0 mL internal std soln 
into flask, stopper, and shake thoroly 10 min. Let settle, filter 
thru Whatman No. 54 paper into g-s flask, and use filtrate for 
analysis. Similarly, prep, soln without internal std, using sam- 
ple contg ca 0.1 g permethrin/25 mL MIBK. 

(c) Emulsifiable concentrates. — Proceed as above under 
wettable and dustable powders. 

(d) Water -dispersible granules. — Grind ca 20 g sample to 
fine powder and thoroly mix. Accurately weigh, in duplicate, 
sample contg 0. 1 g permethrin into 100 mL g-s erlenmeyer. 
Pipet 25 mL internal std soln into flask and place in ultrasonic 
bath 10 min. Proceed as above under wettable and dustable 
powders, beginning "Let settle. . .". 

E. System Performance Check 

Using instrument conditions listed under Apparatus and Re- 
agents, inject 3 or more 1.5 fxL portions of soln CA onto col- 



168 



Pesticide Formulations 



AOAC Official Methods of Analysis (1990) 



umn and adjust paramenters to give peak ht ca 75% full scale 
with peak quality and elution time specified. On-column in- 
jection is necessary. Inject 1.5 u,L solns I, CO, and SO and 
check for interfering peaks. 

F. Determination 

Inject 1.5 jjlL std solns CA and CB until response ratio (total 
area of cis- and /rtws-permethrin peaks /area internal std peak) 
varies <0.5% of mean. (Area measurements by digital elec- 
tronic integration are preferred over other methods.) Carry out 
injection of std and sample solns in following sequence: CA], 
SA 1; SA 2 , CBj, CA 2 , SB), SB 2 , CB 2 . Average response ratios 
for sample and stds that bracket each sample. Successive re- 
sponse ratios should agree ± 5% of their mean. If not, repeat 
analysis. 

Permethrin, % - {R/R') X (W /W) X P 

where R and R' = av. peak area ratios for sample and std, 
resp.; W = g permethrin in std soln; W = g sample extd for 
analysis; and P = % purity of std. 

Ref.: JAOAC 70, 53(1987). 

CAS-52645-53-1 (permethrin) 



938.01 Rotenone in Derris 

and Cube Powder 

Crystallization Method 
Final Action 

{Caution: See safety notes on carbon tetrachloride and toxic 
dusts.) 

A. Reagents 

(a) Purified rotenone. — Dissolve rotenone in boiling CC1 4 ; 
cool in refrigerator or ice bath at 0-10° until pptn of rotenone- 
CC1 4 solvate stops. Filter thru buchner and wash once or twice 
with ice-cold CC1 4 . Cone, filtrate, crystallize, and filter as be- 
fore. Transfer cryst. residue to beaker, add ca twice their vol. 
alcohol, and heat nearly to boiling. (Crystals need not dissolve 
completely.) Cool to room temp., filter thru buchner, and draw 
air thru cryst. residue until most alcohol is removed. Remove 
rotenone from funnel, dry in air, and finally heat 1 hr at 105°. 
Mp, detd in Pyrex, of purified material should be 163-164°. 
(Mother liquors may be coned and rotenone-CCl 4 solvate al- 
lowed to crystallize. Cryst. material may be used for further 
purification, or kept for prepn of wash solns or for seeding to 
induce crystn in detn.) 

(b) Rotenone-CCl 4 solvate. — Ppt rotenone from CC1 4 soln, 
filter by suction, and dry in air, 

(c) Rotenone-CCl 4 wash soln. — Sat. CC1 4 at 0°, and keep 
at 0° during use. 

(d) Alcohol saturated with rotenone at room temp. 

(e) Charcoal, activated. — Norit-A neutral, or equiv. 

B. Preparation of Solution 

(a) Weigh 30 g (if sample contains >7% rotenone, use amt 
to give 1.0—1.5 g rotenone in 200 mL aliquot) finely powd 
root and 10 g of the C, (e), into 500 mL g-s erlenmeyer. Add 
300 mL CHC1 3 , measured at known room temp.; fasten stop- 
per securely and place flask on shaking machine. Agitate vig- 
orously ^4 hr, preferably interrupting shaking with overnight 
rest (or flask may be shaken continuously overnight). Rapidly 
filter mixt. into suitable flask, using fluted paper without suc- 
tion and keeping funnel covered with watch glass to avoid evapn 
loss. Stopper flask and adjust temp, of filtrate to that of orig- 
inal CHCI3. 



(b) Alternative extraction method. — If sample has ratio of 
rotenone to total ext of >0.4, use amt contg 1.0-1.5 g rote- 
none and successively ext 4 times with CHCI3, using 300 mL 
CHCI3 and 4 hr agitation for first extn as in (a) and 200 mL 
and 2 hr each for other extns. Filter after each extn and return 
marc to flask for extn with fresh sol v. Finally combine exts, 
evap. almost to dryness, and use entire ext to det. rotenone. 

(c) Extraction method for formulations containing 0.75-1.0% 
rotenone with or without sulfur and /or pyrethrins. — Weigh 
two 50 g portions sample into sep. 500 mL g-s erlenmeyers. 
Add 5 g of the C and 300 mL CHC1 3 , measured at known 
room temp., to each. Stopper and continue as in (a). 

C Determination 

{Caution: See safety notes on distillation, pipets, acetone, car- 
bon tetrachloride, and chloroform.) 

Pipet 200 mL soln, 938. 01B (or entire soln if alternative 
extn, (b), is used), into 500 mL Pyrex erlenmeyer and distil 
until ca 25 mL remains. (For formulations, 938.01B(c): In ab- 
sence of S , combine the 2 exts in one of the erlenmeyers . In 
presence of S, remove all CHC1 3 on steam bath in air current, 
avoiding prolonged heating. Add 35 mL acetone to each res- 
idue and boil gently on steam bath to dissolve all resins. Re- 
move from steam bath, stopper tightly, and hold 2 hr at 0-5°. 
Filter both acetone solns thru same 15 mL, medium porosity, 
fritted glass buchner into single 500 mL erlenmeyer. Rinse and 
wash with acetone at 5°. Remove acetone as CHC1 3 was re- 
moved above.) 

Evap. almost to dryness on steam bath in current of air. 
Remove remainder of solv. under reduced pressure, heating 
cautiously on steam bath when necessary to hasten evapn. 
(Suction may be applied directly to flask if stopper with vent 
is used to release pressure, so that excessive vac, may be 
avoided. Use flasks with slightly convex bottoms; do not use 
flasks below av. wt,) Dissolve ext in 15 mL hot CC1 4 and 
again, in similar manner, remove all solv. Repeat with another 
10-15 mL portion hot CC1 4 . (This treatment removes all CHC1 3 
from resins. CHC1 3 ext is usually completely sol. in CC1 4 ; if 
small amts of insol. material are present, purification de- 
scribed later will eliminate them.) 

Dissolve residue in ca 10 mL CC1 4 and transfer quant, with 
hot CC1 4 to 50 mL erlenmeyer marked at 25 mL. Adjust vol. 
to 25 mL by evapg on steam bath or by adding CC1 4 . Cool 
flask in ice bath several min, stopper flask, and swirl until 
crystn is apparent. Seed with few crystals of rotenone-CCl 4 
solvate if necessary to induce crystn. If at this stage only small 
amt of cryst. material seps, add accurately weighed amt of 
purified rotenone, 938.01A(a), estd to be enough to assure that 
final result, expressed as pure rotenone, is ^1 g. Then warm 
to dissolve completely, and again induce crystn. At same time 
prep, satd soln of rotenone in CC1 4 , 938.01A(c), for washing. 
Place flasks contg ext and washing soln in ice bath capable of 
holding temp, at 0°, and let stand overnight. (Store ice bath 
in refrigerator to keep ice from melting too rapidly.) 

After 17-18 hr in ice bath, rapidly filter ext thru weighed 
gooch fitted with filter paper disk, removing flask from ice 
bath only long enough to pour each fraction of ext into cru- 
cible. Rinse cryst. residue from flask and wash under suction 
once with the ice-cold satd rotenone-CCl 4 wash soln. (<12— 
15 mL soln should be used for rinsing and washing.) Continue 
suction ca 5 min; then dry to const wt at 40° (ca 1 hr). Wt 
obtained is crude rotenone-CCl 4 solvate. 

Break up contents of crucible with spatula, mix thoroly, and 
weigh 1.000 g into 50 mL erlenmeyer. Add 10 mL alcohol 
previously satd with rotenone at room temp., swirl flask few 
min, stopper tightly, and set aside ^4 hr, preferably overnight, 



AOAC Official Methods of Analysis (1990) 



Natural Products and Synergists 1 69 



at same temp. Filter on weighed gooch fitted with filter paper 
disk, Rinse crystals from flask and wash under suction with 
alcohol satd with rotenone at temp, of recrystn (ca 10 mL usu- 
ally required). Continue suction 3-5 min and then dry crucible 
at 105° to const wt (ca 1 hr). 

Multiply g residue by g total crude rotenone-CCl 4 solvate, 
and add 0.07 g to product as correction for rotenone held in 
soln in the 25 mL CC1 4 used in crystn. If any pure rotenone 
was added, subtract its wt from value obtained. This gives wt 
pure rotenone contained in aliquot of ext. 

Note: Most important precaution in using this method is to 
keep temp, of CCl 4 -rotenone wash soln and crucibles as near 
0° as possible. Keep wash soln surrounded by crushed ice ex- 
cept when actually being used. In warm weather keep cruci- 
bles in refrigerator until ready to use. 

Refs.: Ind. Eng. Chem. Anal. Ed. 10, 19(1938). JAOAC 21, 
148(1938); 22, 408(1939); 24, 70(1941); 43, 376(1960). 

CAS-83-79-4 (rotenone) 



961.03 Rotenone in Derris and Cube Powder 

Infrared Spectroscopic Method 
First Action 

(Not applicable to derris products) 

A. Standardization 

Prep, std solns of purified rotenone, 938.01 A(a), in CHC1 3 
at concns of 5, 10, 15, and 20 mg/mL. Scan each std soln 
from 7.0 to 8.0 fxm at speed of 6 min/|xm and scale of 10 
cm/|xm, using 0.1 mm cell and accurately matching cell filled 
with CHC1 3 as ref. Scan each in duplicate. Obtain av. A of 
each concn, using 7.57 |mm as base point and 7.65 (Jim as peak. 
Plot A against concn. 

B. Determination 

Weigh sample contg 250-300 mg rotenone into 25 x 200 
mm culture tube. Add 1-2 g anhyd. Na 2 S0 4 , 2 g activated 
charcoal, and 50 mL CHC1 3 by pipet. Close securely with Tef- 
lon-lined screw cap and tumble end over end 1 hr at ca 35 
rpm. Filter thru medium paper, avoiding evapn losses. Trans- 
fer 20 mL aliquot to 50 mL erlenmeyer and evap. on steam 
bath with current of air. Transfer residue to 10 mL g-s vol. 
flask and dil. to vol. with CHC1 3 . Stopper, and mix thoroly. 

Scan from 7.0 to 8.0 |xm, using 0.1 mm cell and matched 
cell filled with CHC1 3 as ref. Det. A by baseline method from 
7.57 to 7.75 )xm and peak at 7.65 |xm, using same scanning 
speed and scale expansion as in stdzn. 

Calc. % rotenone from std curve and wt sample in final diln. 

Refs.: JAOAC 44, 580(1961); 46, 668(1963); 59, 380(1976). 
CAS-83-79-4 (rotenone) 



940.04 



Ether Extract of Derris 
and Cube Powder 

Final Action 



flam- 



(Caution: See safety notes on monitoring equipment, 
mable solvents, diethyl ether, and peroxides.) 

Ext 5 g finely powd root with ether 48 hr in Soxhlet or other 
efficient extn app. Cone, ext and filter off any insol. material 
present. Receive filtrate in tared beaker, evap. ether on steam 
bath, and dry in oven at 105° to const wt. 



983.06 Rotenone in Pesticide Formulations 
Liquid Chromatographic Method 
First Action 1983 

A. Principie 

Sample is extd with dioxane, and rotenone is detd by reverse 
phase LC with UV detection at 280 nm. 

B. Apparatus and Reagents 

(a) Liquid chromato graph. — M6000A pump, U6K injector, 
Model 450 variable UV detector (all Waters Associates, Inc.), 
and Omni- Scribe recorder (Houston Instrument, 8500 Cam- 
eron Rd, Austin, TX 78753), or equiv. system. Operating con- 
ditions: column ambient; flow rate 1.0 mL/min for Partisil 
column, 1.5 mL/min for Zorbax column, 1.2 mL/min for 
Bondapak column; injection vol. 5 pJL for Partisil column and 
10 |xL for others; detector wavelength 280 nm; absorbance range 
0.4 AUFS; chart speed 1 cm/min. 

(b) Chromatographic columns. — Partisil 5 ODS-3, 5 fxm 
particle size, stainless steel, 25 cm X 4.6 mm id (Whatman 
Inc.). Zorbax C 8 , 10 \xm particle size, stainless steel, 25 cm 
x 4.6 mm id (DuPont Co.). jxBondapak C 18 , 10 \xm particle 
size, stainless steel, 30 cm X 3.9 mm id (Waters Associates, 
Inc.). 

(c) Mobile phases. — Use LC grade org. solvs (Fisher Sci- 
entific Co.). Use glass-distd H 2 treated to remove org. compds 
by passing thru C 18 column system (Millipore Corp.) or use 
LC grade H 2 0. Use MeOH-H 2 (75 + 25), (68+32), and 
(66+34) for Partisil, Zorbax, and Bondapak, resp. If neces- 
sary, adjust mobile phase to give adequate sepn of tephrosin, 
rotenone, and deguelin in test soln (Fig. 983.06). 

(d) Test soln. — Accurately weigh portion of well mixed 
sample of Noxfish Fish Toxicant or powd cube root ext (Rous- 
sel BioCorp, 400 Sylvan Ave, PO Box 1077, Lyndhurst, NJ 
07071) contg ca 20 mg rotenone into 125 mL g-s erlenmeyer. 
Add 50 mL dioxane, and mix. 

(e) Std soln. — Accurately weigh ca 20 mg 99% pure rote- 
none (Penick Co.) into 50 mL vol. flask and dil. to vol. with 



w 



_JUU 




10 



12 



14 



TIME(min) 



FIG. 983.06 — Liq. chromatogram of rotenone sample with 
Whatman column: a, rotenolone; b, tephrosin and an un- 
known; c, rotenone; d, deguelin 



170 



Pesticide Formulations 



AOAC Official Methods of Analysis (1990) 



dioxane (reagent grade). Keep rotenone from light or store in 
actinic glassware. 

(f) Sample extraction solvent. — Reagent grade dioxane. 

C. Preparation of Sample 

(a) Solid formulations. — Accurately weigh portion of well 
mixed sample contg ca 20 mg rotenone into 125 mL g-s er- 
lenmeyer. Pipet in 50 mL dioxane, stopper, and shake 1 i / 2 hr 
on rotary shaker. Let settle and filter aliquot thru 0.45 |xm 
organic filter (Millipore Corp.), or equiv. 

(b) Liquid formulations. — Use same procedure as above, 
omitting rotary shaking and settling. 

D. Determination and Calculation 

Inject std soln followed by 2 injections of sample soln and 
another injection of std soln. Measure peak hts, average, and 
calc. as follows: 

% Rotenone - (PH/PH') X (W /W) X % purity of std 

where PH and PM' = av. peak hts of sample and std solns, 
resp.; W = g rotenone std/50 mL; and W - g sample extd. 

Ref.: J AOAC 66, 796(1983). 

CAS-83-79-4 (rotenone) 



960.11 Piperonyl Butoxide 

in Pesticide Formulations 
Colorimetric Method 

First Action 1960 
Final Action 1961 

A. Apparatus and Reagents 

(a) Photoelectric colorimeter. — Equipped with narrow band- 
pass interference type filter with central wavelength 630 nm. 
(Filter is available from PTR Optics Corp, 145 Newton St, 
Waltham, MA 02154.) Spectrophtr set at wavelength in range 
625-635 nm may also be used. 

(b) Purified tannic acid. — Purify as follows: To 20 g tannic 
acid (USP reagent grade) add 100 mL EtOAc (99%) and stir 
mech. ca 1 hr. Filter by suction thru fritted glass funnel, and 
wash residue with three 5 mL portions EtOAc. To combined 
filtrate and washings add 2 g finely powd Darco G-60 (or equiv. 
decolorizing C), and stir mech. ca 0.5 hr. Filter by gravity 
thru double thickness Whatman No. 1, or equiv., paper into 
graduated dropping funnel. Wash residue several times with 
EtOAc until vol. filtrate and washings is ca 125 mL. Place 
dropping funnel over 1 L, 3 -neck, r-b flask, equipped with 
mech. stirrer, and with vigorous agitation in flask, add filtrate 
dropwise to 5 times its vol. of toluene. Purified tannic acid is 
pptd immediately. 

Filter by suction thru fritted glass funnel, and wash product 
thoroly with toluene, stirring solids with toluene to assure 
complete removal of EtOAc. Continue suction until practically 
all toluene is removed. Dry purified tannic acid in vac. oven 
at ca 40°, and place in tightly stoppered bottle. 

(c) Tannic acid reagent. — Completely dissolve exactly 0.025 
g purified tannic acid in 20 mL HOAc by shaking at room 
temp. Add 80 mL H 3 P0 4 and mix thoroly. Prep, fresh daily. 
Store tightly stoppered, as it is hygroscopic. 

(d) Purified piperonyl butoxide. — Purify by low pressure 
fractional distn of tech. product. (Caution: See safety notes on 
vacuum.) Also available from Fairfield American Corp., 3932 
Salt Rd, Medina, NY 14103. 

(e) Piperonyl butoxide std soln. — 50 juLg/0. 1 mL. Weigh 
exactly 1.000 g purified piperonyl butoxide into 100 mL vol. 
flask. (Hypodermic syringe and needle are convenient for add- 



ing compd to flask.) Dil. to vol. with deodorized kerosene and 
mix well. Pipet 10 mL of this soln into 200 mL vol. flask. 
Dil. to vol. with deodorized kerosene and mix well. This soln 
is stable for several months. If std is to be used with sample 
contg pyrethrum, add enough pyrethrum ext to std before ini- 
tial diln to give ratio piperonyl butoxide to pyrethrins similar 
to sample. 

B. Preparation of Sample 

Accurately weigh sample contg 0.5-1.5 g piperonyl butox- 
ide into tared 100 mL vol. flask, dil. to vol. with deodorized 
kerosene, and mix well. Pipet 10 mL into 200 mL vol. flask, 
dil. to vol. with deodorized kerosene, and mix well. 

C. Determination 

Pipet 0.1 mL (from 1 mL pipet graduated in 0.1 mL) sample 
soln into 18 x 150 mm test tube. Add exactly 5 mL tannic 
acid reagent and shake vigorously 1 min. Treat std and blank, 
consisting of 0. 1 mL deodorized kerosene, simultaneously in 
same manner. 

Place test tubes in test-tube basket and place in vigorously 
boiling H 2 bath 5 min. Remove basket and let tubes cool to 
room temp. Transfer solns to colorimeter tubes and read, against 
H 2 0, using 625-635 nm filter or setting. (After cooling to 
room temp, there is no appreciable change in A for several 
hr.) 

Subtract A of deodorized kerosene from readings of both 
sample, A, and std, A f . 

mg Piperonyl butoxide = A x 0.05 /A' 

Refs.: JAOAC 35, 771(1952); 43, 350(1960). 

CAS-5 1-03-6 (piperonyl butoxide) 



936.05 Pyrethrin in Pesticide Formulations 
Mercury Reduction Method (1) 
Final Action 

(Caution: See safety notes on flammable solvents, diethyl ether, 
peroxides, and petroleum ether.) 

A. Reagents 

(a) Deniges reagent. — Mix 5 g yellow HgO with 40 mL 
H 2 0, and, while stirring, slowly add 20 mL H 2 S0 4 ; then add 
addnl 40 mL H 2 and stir until all